Treatment of a disease of the gastrointestinal tract with a TLR modulator

ABSTRACT

This disclosure features methods and compositions for treating diseases of the gastrointestinal tract with a TLR agonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. § 371of International Application No. PCT/US2017/066459, filed Dec. 14, 2017,which claims the benefit of priority to the following U.S. ProvisionalApplications 62/434,366 filed Dec. 14, 2016; 62/478,840 filed Mar. 30,2017; 62/545,219 filed Aug. 14, 2017; and 62/583,800 filed Nov. 9, 2017,each of which is incorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure features methods and compositions for treating diseasesof the gastrointestinal tract with a TLR modulator.

BACKGROUND

Toll-like receptor 9 (TLR9, also knowns as CD289 (cluster ofdifferentiation 289)) is a member of the toll-like receptor (TLR)family. TLR9 is present inside immune cells, as well as on the surfaceof epithelial cells. TLR9 has been implicated in progression of Crohn'sdisease and ulcerative colitis.

The gastrointestinal (GI) tract generally provides a therapeutic mediumfor an individual's body. At times, therapeutic drugs may need to bedispensed to specified locations within the small intestine or largeintestine, which is more effective than oral administration of thetherapeutic drugs to cure or alleviate the symptoms of some medicalconditions. For example, therapeutic drugs dispensed directly within thesmall intestine would not be contaminated, digested or otherwisecompromised in the stomach, and thus allow a higher dose to be deliveredat a specific location within the small intestine. However, dispensingtherapeutic drugs directly within the small intestine inside a humanbody (e.g., the cecum, the ascending colon) can be difficult, because adevice or mechanism (e.g., special formulation) would be needed totransport a therapeutically effective dose of drug to a desired locationwithin the small intestine and then automatically deliver thetherapeutic drug at the desired location. Dispensing therapeutic drugsdirectly within other locations in the GI tract of the human body can besimilarly difficult. Such a device or mechanism also would also need tobe operated in a safe manner in that the device or mechanism needs tophysically enter the human body.

In sum, there remains a significant unmet medical need for improvedtreatment regimens for gastrointestinal diseases, such as inflammatorybowel disease (IBD), including a need for regimens which can dispensetherapeutics to specific locations within the GI tract, thereby reducingor avoiding the drawbacks of oral or other forms of systemicadministration.

SUMMARY

The present disclosure provides novel treatment paradigms forinflammatory conditions of the gastrointestinal tract. The methods andcompositions described herein allow for the regio-specific release oftherapeutic drugs at or near the site of disease in the gastrointestinaltract. By releasing a therapeutic drug locally instead of systemically,the bioavailability of the drug can be increased at the site of injuryand/or decreased in the systemic circulation, thereby resulting inimproved overall safety and/or efficacy and fewer adverse side effects.Advantages may include one or more of increased drug engagement at thetarget, leading to new and more efficacious treatment regimens, and/orlower systemic drug levels, which can translate to reduced toxicity andreduced immunogenicity, e.g., in the case of biologics. In someinstances, releasing a therapeutic drug locally also provides for newmodes of action that may be unique to local delivery in the GI tract asopposed to systemic administration. For patients, clinicians and payors,this can mean an easier or simpler route of administration, fewerco-medicaments (e.g., immunomodulators), fewer side effects, and/orbetter outcomes.

Accordingly, described herein are methods for treating disorders of thegastrointestinal (GI) tract. The methods can include one or more of:

-   -   diagnosing a GI disease in a subject; and/or    -   mapping, sampling, and/or assessing the site, severity,        pathology, and extent of a GI disease in the GI tract of a        subject and/or mapping, sampling, and/or assessing a patient        response to a therapeutic agent, e.g., in the patient's GI        tract; and/or    -   identifying, quantifying, and/or monitoring one or more markers        of a GI disease in the GI tract of the subject and/or one or        more markers of patient response to a therapeutic agent, e.g.,        in the patient's GI tract; and/or    -   releasing a therapeutic agent, e.g., proximate to the site of a        GI disease.

The present disclosure accordingly provides patients and physicians morepersonalized treatment options for GI disorders by facilitating regimenswhich can release a therapeutic agent according to desired (e.g.,customized or optimized) dosage, timing, and/or location parameters. Insome cases, the treatment methods can employ one or more ingestibledevices to achieve the benefits disclosed herein.

In some embodiments, provided herein is a method of treating a diseaseof the gastrointestinal tract in a subject, comprising:

administering to the subject a pharmaceutical formulation that comprisesan TLR agonist,

wherein the pharmaceutical formulation is released at a location in thegastrointestinal tract of the subject that is proximate to one or moresites of disease.

In some embodiments, provided herein the pharmaceutical formulation isadministered in an ingestible device. In some embodiments, thepharmaceutical formulation is released from an ingestible device. Insome embodiments, the ingestible device comprises a housing, a reservoircontaining the pharmaceutical formulation, and a release mechanism forreleasing the pharmaceutical formulation from the device,

wherein the reservoir is releasably or permanently attached to theexterior of the housing or internal to the housing.

In some embodiments, provided herein is a method of treating a diseaseof the gastrointestinal tract in a subject, comprising:

administering to the subject an ingestible device comprising a housing,a reservoir containing a pharmaceutical formulation, and a releasemechanism for releasing the pharmaceutical formulation from the device,

wherein the reservoir is releasably or permanently attached to theexterior of the housing or internal to the housing;

wherein the pharmaceutical formulation comprises an TLR agonist, and

the ingestible device releases the pharmaceutical formulation at alocation in the gastrointestinal tract of the subject that is proximateto one or more sites of disease.

In some embodiments, the housing is non-biodegradable in the GI tract.In some embodiments, the release of the formulation is triggeredautonomously. In some embodiments, the device is programmed to releasethe formulation with one or more release profiles that may be the sameor different at one or more locations. In some embodiments, the deviceis programmed to release the formulation at a location proximate to oneor more sites of disease. In some embodiments, the location of one ormore sites of disease is predetermined.

In some embodiments, the reservoir is made of a material that allows theformulation to leave the reservoir, such as a biodegradable material.

In some embodiments, the release of the formulation is triggered by apre-programmed algorithm. In some embodiments, the release of theformulation is triggered by data from a sensor or detector to identifythe location of the device. In some more particular embodiments, thedata is not based solely on a physiological parameter (such as pH,temperature, and/or transit time).

In some embodiments, the device comprises a detector configured todetect light reflectance from an environment external to the housing. Insome more particular embodiments, the release is triggered autonomouslyor based on the detected reflectance.

In some embodiments, the device releases the formulation atsubstantially the same time as one or more sites of disease aredetected. In some embodiments, the one or more sites of disease aredetected by the device (e.g., by imaging the GI tract).

In some embodiments, the release mechanism is an actuation system. Insome embodiments, the release mechanism is a chemical actuation system.In some embodiments, the release mechanism is a mechanical actuationsystem. In some embodiments, the release mechanism is an electricalactuation system. In some embodiments, the actuation system comprises apump and releasing the formulation comprises pumping the formulation outof the reservoir. In some embodiments, the actuation system comprises agas generating cell. In some embodiments, the device further comprisesan anchoring mechanism. In some embodiments, the formulation comprises atherapeutically effective amount of the TLR agonist. In someembodiments, the formulation comprises a human equivalent dose (HED) ofthe TLR agonist.

In some embodiments, the device is a device capable of releasing a solidTLR agonist or a solid formulation comprising the TLR agonist. In someembodiments, the device is a device capable of releasing a liquid TLRagonist or a liquid formulation comprising the TLR agonist. Accordingly,in some embodiments of the methods herein, the pharmaceuticalformulation release from the device is a solid formulation. Accordingly,in some embodiments of the methods herein, the pharmaceuticalformulation release from the device is a liquid formulation.

The devices disclosed herein are capable of releasing a TLR agonist or aformulation comprising the TLR agonist irrespective of the particulartype of TLR agonist. For example, the TLR agonist may be a smallmolecule, a biological, a nucleic acid, an antibody, a fusion protein,and so on.

In some embodiments, provided herein is a method of releasing an TLRagonist into the gastrointestinal tract of a subject for treating one ormore sites of disease within the gastrointestinal tract, the methodcomprising:

administering to the subject a therapeutically effective amount of theTLR agonist housed in an ingestible device, wherein the ingestibledevice comprises

a detector configured to detect the presence of the one or more sites ofdisease, and

a controller or processor configured to trigger the release of the TLRagonist proximate to the one or more sites of disease in response to thedetector detecting the presence of the one or more sites of disease.

In some embodiments, provided herein is a method of releasing an TLRagonist into the gastrointestinal tract of a subject for treating one ormore pre-determined sites of disease within the gastrointestinal tract,the method comprising:

administering to the subject a therapeutically effective amount of theTLR agonist contained in an ingestible device, wherein the ingestibledevice comprises

a detector configured to detect the location of the device within thegastrointestinal tract, and

a controller or processor configured to trigger the release of the TLRagonist proximate to the one or more predetermined sites of disease inresponse to the detector detecting a location of the device thatcorresponds to the location of the one or more pre-determined sites ofdisease.

In some embodiments, provided herein is a method of releasing an TLRagonist into the gastrointestinal tract of a subject for treating one ormore sites of disease within the gastrointestinal tract, the methodcomprising:

administering to the subject a therapeutically effective amount of theTLR agonist contained in an ingestible device;

receiving at an external receiver from the device a signal transmittingenvironmental data;

assessing the environmental data to confirm the presence of the one ormore sites of disease; and

when the presence of the one or more sites of disease is confirmed,sending from an external transmitter to the device a signal triggeringthe release of the TLR agonist proximate to the one or more sites ofdisease.

In some embodiments, provided herein is a method of releasing an TLRagonist into the gastrointestinal tract of a subject for treating one ormore sites of disease within the gastrointestinal tract, the methodcomprising:

administering to the subject a therapeutically effective amount of theTLR agonist contained in an ingestible device;

receiving at an external receiver from the device a signal transmittingenvironmental or optical data;

assessing the environmental or optical data to confirm the location ofthe device within the gastrointestinal tract; and

when the location of the device is confirmed, sending from an externaltransmitter to the device a signal triggering the release of the TLRagonist proximate to the one or more sites of disease.

Provided herein in one embodiment is a method of treating a disease ofthe gastrointestinal tract in a subject, comprising:

delivering a TLR modulator at a location in the gastrointestinal tractof the subject, wherein the method comprises administering to thesubject a pharmaceutical composition comprising a therapeuticallyeffective amount of the TLR modulator.

Provided herein in one embodiment is a method of treating a disease ofthe large intestine in a subject, comprising:

delivering a TLR modulator at a location in the proximal portion of thelarge intestine of the subject,

wherein the method comprises administering endoscopically to the subjecta therapeutically effective amount of the TLR modulator.

Provided herein in one embodiment is a method of treating a disease ofthe gastrointestinal tract in a subject, comprising:

releasing a TLR modulator at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of disease,

wherein the method comprises administering to the subject apharmaceutical composition comprising a therapeutically effective amountof the TLR modulator.

Provided herein in one embodiment is a method of treating a disease ofthe gastrointestinal tract in a subject, comprising:

releasing a TLR modulator at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of disease,

wherein the method comprises administering to the subject apharmaceutical composition comprising a therapeutically effective amountof the TLR modulator, wherein the pharmaceutical composition is aningestible device. and the method comprises administering orally to thesubject the pharmaceutical composition.

Provided herein in one embodiment is a method of treating a disease ofthe gastrointestinal tract in a subject, comprising:

releasing a TLR modulator at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of disease, whereinthe method comprises administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of the TLRmodulator, wherein the method provides a concentration of the TLRmodulator in the plasma of the subject that is less than 3 μg/ml.

Provided herein in one embodiment is a method of treating a disease ofthe large intestine in a subject, comprising:

releasing a TLR modulator at a location in the proximal portion of thelarge intestine of the subject that is proximate to one or more sites ofdisease,

wherein the method comprises administering endoscopically to the subjecta therapeutically effective amount of the TLR modulator.

In another aspect of the present invention, there is provided a TLRmodulator for use in a method of treating a disease of thegastrointestinal tract in a subject, wherein the method comprises orallyadministering to the subject an ingestible device loaded with the TLRmodulator, wherein the TLR modulator is released by the device at alocation in the gastrointestinal tract of the subject that is proximateto one or more sites of disease.

In another aspect, the present invention provides a compositioncomprising or consisting of an ingestible device loaded with atherapeutically effective amount of a TLR modulator, for use in a methodof treatment, wherein the method comprises orally administering thecomposition to the subject, wherein the TLR modulator is released by thedevice at a location in the gastrointestinal tract of the subject thatis proximate to one or more sites of disease.

In another aspect, the present invention provides an ingestible deviceloaded with a therapeutically effective amount of a TLR modulator,wherein the device is controllable to release the TLR modulator at alocation in the gastrointestinal tract of the subject that is proximateto one or more sites of disease. The device may be for use in a methodof treatment of the human or animal body, for example, any method asdescribed herein.

In still another aspect, the present invention provides an ingestibledevice for use in a method of treating a disease of the gastrointestinaltract in a subject, wherein the method comprises orally administering tothe subject the ingestible device loaded with a therapeuticallyeffective amount of a TLR modulator, wherein the TLR modulator isreleased by the device at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of disease.

An ingestible device as used in the present invention may comprise oneor more mechanical and/or electrical mechanisms which actively controlrelease of the TLR modulator. For example, in any of the above aspectsand embodiments, the ingestible device as used in the present inventionmay comprise a release mechanism for release of the TLR modulator (e.g.,from a reservoir comprising the TLR modulator) and an actuatorcontrolling the release mechanism.

In one embodiment, the ingestible device comprises:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR modulator stored therein;

a release mechanism having a closed state which retains the TLRmodulator in the reservoir and an open state which releases the TLRmodulator from the reservoir to the exterior of the device; and

an actuator which changes the state of the release mechanism from theclosed to the open state.

In one embodiment, the ingestible device comprises:

a housing defined by a first end, a second end substantially oppositefrom the first end;

a reservoir located within the housing and containing the TLR modulatorwherein a first end of the reservoir is attached to the first end of thehousing;

a mechanism for releasing the TLR modulator from the reservoir;

and

an exit valve configured to allow the TLR modulator to be released outof the housing from the reservoir.

Here, the exit valve can be considered as the release mechanism having aclosed state which retains the TLR modulator in the reservoir and anopen state which releases the TLR modulator from the reservoir to theexterior of the device, and the mechanism for releasing the TLRmodulator from the reservoir can be considered as the actuator.

In some embodiments of methods of treatment as described herein, the oneor more disease sites may have been pre-determined (e.g., determined ina step preceding the administration of the composition of the presentinvention). The disease site(s) may have been determined by imaging thegastrointestinal tract. For example, the disease site(s) may have beenpre-determined by endoscopy (e.g., a step of colonoscopy, enteroscopy,or using a capsule endoscope). Determination that the device isproximate to the disease site may therefore comprise a determining thatthe device is in a location corresponding to this previously-determineddisease site.

In some embodiments, the location of the device in the gut may bedetected by tracking the device. For example, the device may comprise alocalization mechanism which may be a communication system fortransmitting localization data, e.g., by radiofrequency transmission.The device may additionally or alternatively comprise a communicationsystem for receiving a signal remotely triggering the actuator and thuscausing release of the TLR modulator. The signal may be sent when it isdetermined that the device is in the correct location in the gut.

Thus, the ingestible device may comprise:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR modulator stored therein;

a release mechanism having a closed state which retains the TLRmodulator in the reservoir and an open state which releases the TLRmodulator from the reservoir to the exterior of the device;

a communication system for transmitting localization data to an externalreceiver and for receiving a signal from an external transmitter; and

an actuator which changes the state of the release mechanism from theclosed to the open state and which can be triggered by the signal.

In other embodiments, the ingestible device as used in the presentinvention may comprise an environmental sensor for detecting thelocation of the device in the gut and/or for detecting the presence ofdisease in the GI tract. For example, the environment sensor may be animage sensor for obtaining images in vivo.

Detecting the presence of disease may comprise, for example, detectingthe presence of inflamed tissue, and/or lesions such as ulceration e.g.,aphthoid ulcerations, “punched-out ulcers” and/or superficial ulcers ofthe mucosa, cobblestoning, stenosis, granulomas, crypt abscesses,fissures, e.g., extensive linear fissures, villous atrophy, fibrosis,and/or bleeding.

Detecting the presence of disease may also comprise molecular sensing,such as detecting the amount of an inflammatory cytokine or other markerof inflammation. Such a marker can be measured locally from a biopsy orsystemically in the serum.

Where the ingestible device comprises an environmental sensor, actuationof the release mechanism may be triggered by a processor or controllercommunicably coupled to the environmental sensor. Thus, in someembodiments, the device may not require any external signal or controlin order to release the drug.

In one embodiment, the ingestible device may comprise:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR modulator stored therein;

a release mechanism having a closed state which retains the TLRmodulator in the reservoir and an open state which releases the TLRmodulator from the reservoir to the exterior of the device;

an actuator which controls the transition of the release mechanism fromthe closed to the open state;

a detector for detecting the location of the device in the gut and/orthe presence of diseased tissue; and

a processor or controller which is coupled to the detector and to theactuator and which triggers the actuator to cause the release mechanismto transition from its closed state to its open state when it isdetermined that the device is in the presence of diseased tissue and/orin a location in the gut that has been predetermined to be proximal todiseased tissue.

In another embodiment, there is provided:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR modulator stored therein;

a detector coupled to the ingestible housing, the detector configured todetect when the ingestible housing is proximate to a respective diseasesite of the one of the one or more sites of disease;

a valve system in fluid communication with the reservoir system; and

a controller communicably coupled to the valve system and the detector,the controller configured to cause the valve system to open in responseto the detector detecting that the ingestible housing is proximate tothe respective disease site so as to release the therapeuticallyeffective amount of the TLR modulator at the respective disease site.

As above, detection that the ingestible housing is proximate to therespective disease site may be based on environmental data indicatingthe location of the device in the GI tract (and reference to apre-determined disease site) or on environmental data directlyindicating the presence of diseased tissue.

Additionally, or alternatively, the device may further comprise acommunication system adapted to transmit the environment data to anexternal receiver (e.g., outside of the body). This data may be used,for example, for diagnostic purposes. The external receiver may comprisemeans for displaying the data.

In some embodiments, this data may be analyzed externally to the deviceand used to determine when the drug should be released: an externalsignal may then be sent to the device to trigger release of the drug.Thus, the communication system may further be adapted to receive asignal remotely triggering the actuator and thus causing release of theTLR modulator. The signal may be sent from an external transmitter inresponse to receipt/analysis and/or assessment of the environmentaldata, e.g., data indicating that the device has reached the desiredlocation of the gut (where the location of the diseased tissue has beenpre-determined) and/or data indicating the presence of diseased tissue.“External” may be “outside of the body”.

Thus, in another embodiment, the ingestible device may comprise:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR modulator stored therein;

a release mechanism having a closed state which retains the TLRmodulator in the reservoir and an open state which releases the TLRmodulator from the reservoir to the exterior of the device;

an environmental detector for detecting environmental data indicatingthe location of the device in the gut and/or the presence of diseasedtissue;

a communication system for transmitting the environmental data to anexternal receiver and for receiving a signal from an externaltransmitter; and

an actuator which controls the transition of the release mechanism fromthe closed to the open state in response to the signal.

It will be understood from the above that when the device comprises oneor more environmental detectors, e.g., comprises an image detector, thecompositions may be used both for disease detection and for diseasetreatment.

Accordingly, in a further embodiment, there is provided a TLR modulatorfor use in a method of detecting and treating a disease of thegastrointestinal tract in a subject, wherein the method comprises orallyadministering to the subject an ingestible device loaded with the TLRmodulator, wherein the ingestible device comprises an environmentalsensor for determining the presence of diseased tissue in the GI tract,and wherein the TLR modulator is released by the device at a location inthe gastrointestinal tract of the subject that is proximate to one ormore sites of disease, as detected by the environmental sensor. Thedevice may be according to any of the embodiments described herein.

In another embodiment, there is provided a composition for use in amethod of detecting and treating a disease of the gastrointestinal tractin a subject, wherein the composition comprises or consists of aningestible device loaded with a therapeutically effective amount of aTLR modulator, wherein the ingestible device comprises an environmentalsensor for determining the presence of diseased tissue in the GI tract,and wherein the TLR modulator is released by the device at a location inthe gastrointestinal tract of the subject that is proximate to one ormore sites of disease, as detected by the environmental sensor. Again,the device may be according to any of the embodiments described herein.

In some embodiments, where the ingestible device as used in the presentinvention comprises an environmental sensor for detecting the presenceof disease in the GI tract and a communication system as describedabove, the method of treatment may comprise:

i) receiving at an external receiver from the ingestible device a signaltransmitting the environmental data;

ii) assessing the environmental data to confirm the presence of thedisease; and

iii) when the presence of the disease is confirmed, sending from anexternal transmitter to the ingestible device a signal triggeringrelease of the TLR modulator.

For example, the presence of disease may be confirmed based on thepresence of inflamed tissue and/or lesions associated with any of thedisease states referred to herein. For example, the presence of diseasemay be confirmed based on the presence of inflammation, ulceration e.g.,aphthoid ulcerations, “punched-out ulcers” and/or superficial ulcers ofthe mucosa, cobblestoning, stenosis, granulomas, crypt abscesses,fissures, e.g., extensive linear fissures, villous atrophy, fibrosis,and/or bleeding.

In some embodiments, the present invention may relate to a systemcomprising: an ingestible device loaded with a therapeutically effectiveamount of a TLR modulator, a release mechanism for release of the TLRmodulator (e.g., from a reservoir comprising the TLR modulator), anactuator controlling the release mechanism, an environmental sensor fordetermining the location of the device in the gut and/or for detectingthe presence of diseased tissue and a communication system adapted totransmit the environment data and receive a signal triggering theactuator;

a receiver and display module for receiving and displaying outside ofthe body the environment data from the ingestible device;

a transmitter for sending to the ingestible device a signal triggeringthe actuator.

Provided herein in one embodiment is a method of treating a disease ofthe gastrointestinal tract in a subject, comprising:

delivering a TLR agonist at a location in the gastrointestinal tract ofthe subject, wherein the method comprises administering to the subject apharmaceutical composition comprising a therapeutically effective amountof the TLR agonist.

Provided herein in one embodiment is a method of treating a disease ofthe large intestine in a subject, comprising:

delivering a TLR agonist at a location in the proximal portion of thelarge intestine of the subject,

wherein the method comprises administering endoscopically to the subjecta therapeutically effective amount of the TLR agonist.

Provided herein in one embodiment is a method of treating a disease ofthe gastrointestinal tract in a subject, comprising:

releasing a TLR agonist at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of disease,

wherein the method comprises administering to the subject apharmaceutical composition comprising a therapeutically effective amountof the TLR agonist.

Provided herein in one embodiment is a method of treating a disease ofthe gastrointestinal tract in a subject, comprising:

releasing a TLR agonist at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of disease,

wherein the method comprises administering to the subject apharmaceutical composition comprising a therapeutically effective amountof the TLR agonist, wherein the pharmaceutical composition is aningestible device. and the method comprises administering orally to thesubject the pharmaceutical composition.

Provided herein in one embodiment is a method of treating a disease ofthe gastrointestinal tract in a subject, comprising:

releasing a TLR agonist at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of disease, whereinthe method comprises administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of the TLRagonist, wherein the method provides a concentration of the TLR agonistin the plasma of the subject that is less than 3 μg/ml.

Provided herein in one embodiment is a method of treating a disease ofthe large intestine in a subject, comprising:

releasing a TLR agonist at a location in the proximal portion of thelarge intestine of the subject that is proximate to one or more sites ofdisease,

wherein the method comprises administering endoscopically to the subjecta therapeutically effective amount of the TLR agonist.

In another aspect of the present invention, there is provided a TLRagonist for use in a method of treating a disease of thegastrointestinal tract in a subject, wherein the method comprises orallyadministering to the subject an ingestible device loaded with the TLRagonist, wherein the TLR agonist is released by the device at a locationin the gastrointestinal tract of the subject that is proximate to one ormore sites of disease.

In another aspect, the present invention provides a compositioncomprising or consisting of an ingestible device loaded with atherapeutically effective amount of a TLR agonist, for use in a methodof treatment, wherein the method comprises orally administering thecomposition to the subject, wherein the TLR agonist is released by thedevice at a location in the gastrointestinal tract of the subject thatis proximate to one or more sites of disease.

In another aspect, the present invention provides an ingestible deviceloaded with a therapeutically effective amount of a TLR agonist, whereinthe device is controllable to release the TLR agonist at a location inthe gastrointestinal tract of the subject that is proximate to one ormore sites of disease. The device may be for use in a method oftreatment of the human or animal body, for example, any method asdescribed herein.

In still another aspect, the present invention provides an ingestibledevice for use in a method of treating a disease of the gastrointestinaltract in a subject, wherein the method comprises orally administering tothe subject the ingestible device loaded with a therapeuticallyeffective amount of a TLR agonist, wherein the TLR agonist is releasedby the device at a location in the gastrointestinal tract of the subjectthat is proximate to one or more sites of disease.

An ingestible device as used in the present invention may comprise oneor more mechanical and/or electrical mechanisms which actively controlrelease of the TLR agonist. For example, in any of the above aspects andembodiments, the ingestible device as used in the present invention maycomprise a release mechanism for release of the TLR agonist (e.g., froma reservoir comprising the TLR agonist) and an actuator controlling therelease mechanism.

In one embodiment, the ingestible device comprises:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR agonist stored therein;

a release mechanism having a closed state which retains the TLR agonistin the reservoir and an open state which releases the TLR agonist fromthe reservoir to the exterior of the device; and

an actuator which changes the state of the release mechanism from theclosed to the open state.

In one embodiment, the ingestible device comprises

a housing defined by a first end, a second end substantially oppositefrom the first end;

a reservoir located within the housing and containing the TLR agonistwherein a first end of the reservoir is attached to the first end of thehousing;

a mechanism for releasing the TLR agonist from the reservoir;

and

an exit valve configured to allow the TLR agonist to be released out ofthe housing from the reservoir.

Here, the exit valve can be considered as the release mechanism having aclosed state which retains the TLR agonist in the reservoir and an openstate which releases the TLR agonist from the reservoir to the exteriorof the device, and the mechanism for releasing the TLR agonist from thereservoir can be considered as the actuator.

In some embodiments of methods of treatment as described herein, the oneor more disease sites may have been pre-determined (e.g., determined ina step preceding the administration of the composition of the presentinvention). The disease site(s) may have been determined by imaging thegastrointestinal tract. For example, the disease site(s) may have beenpre-determined by endoscopy (e.g., a step of colonoscopy, enteroscopy,or using a capsule endoscope). Determination that the device isproximate to the disease site may therefore comprise a determining thatthe device is in a location corresponding to this previously-determineddisease site.

In some embodiments, the location of the device in the gut may bedetected by tracking the device. For example, the device may comprise alocalization mechanism which may be a communication system fortransmitting localization data, e.g., by radiofrequency transmission.The device may additionally or alternatively comprise a communicationsystem for receiving a signal remotely triggering the actuator and thuscausing release of the TLR agonist. The signal may be sent when it isdetermined that the device is in the correct location in the gut.

Thus, the ingestible device may comprise:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR agonist stored therein;

a release mechanism having a closed state which retains the TLR agonistin the reservoir and an open state which releases the TLR agonist fromthe reservoir to the exterior of the device;

a communication system for transmitting localization data to an externalreceiver and for receiving a signal from an external transmitter; and

an actuator which changes the state of the release mechanism from theclosed to the open state and which can be triggered by the signal.

In other embodiments, the ingestible device as used in the presentinvention may comprise an environmental sensor for detecting thelocation of the device in the gut and/or for detecting the presence ofdisease in the GI tract. For example, the environment sensor may be animage sensor for obtaining images in vivo.

Detecting the presence of disease may comprise, for example, detectingthe presence of inflamed tissue, and/or lesions such as ulceration e.g.,aphthoid ulcerations, “punched-out ulcers” and/or superficial ulcers ofthe mucosa, cobblestoning, stenosis, granulomas, crypt abscesses,fissures, e.g., extensive linear fissures, villous atrophy, fibrosis,and/or bleeding.

Detecting the presence of disease may also comprise molecular sensing,such as detecting the amount of an inflammatory cytokine or other markerof inflammation. Such a marker can be measured locally from a biopsy orsystemically in the serum.

Where the ingestible device comprises an environmental sensor, actuationof the release mechanism may be triggered by a processor or controllercommunicably coupled to the environmental sensor. Thus, in someembodiments, the device may not require any external signal or controlin order to release the drug.

In one embodiment, the ingestible device may comprise:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR agonist stored therein;

a release mechanism having a closed state which retains the TLR agonistin the reservoir and an open state which releases the TLR agonist fromthe reservoir to the exterior of the device;

an actuator which controls the transition of the release mechanism fromthe closed to the open state;

a detector for detecting the location of the device in the gut and/orthe presence of diseased tissue; and

a processor or controller which is coupled to the detector and to theactuator and which triggers the actuator to cause the release mechanismto transition from its closed state to its open state when it isdetermined that the device is in the presence of diseased tissue and/orin a location in the gut that has been predetermined to be proximal todiseased tissue.

In another embodiment, there is provided:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR agonist stored therein;

a detector coupled to the ingestible housing, the detector configured todetect when the ingestible housing is proximate to a respective diseasesite of the one of the one or more sites of disease;

a valve system in fluid communication with the reservoir system; and

a controller communicably coupled to the valve system and the detector,the controller configured to cause the valve system to open in responseto the detector detecting that the ingestible housing is proximate tothe respective disease site so as to release the therapeuticallyeffective amount of the TLR agonist at the respective disease site.

As above, detection that the ingestible housing is proximate to therespective disease site may be based on environmental data indicatingthe location of the device in the GI tract (and reference to apre-determined disease site) or on environmental data directlyindicating the presence of diseased tissue.

Additionally, or alternatively, the device may further comprise acommunication system adapted to transmit the environment data to anexternal receiver (e.g., outside of the body). This data may be used,for example, for diagnostic purposes. The external receiver may comprisemeans for displaying the data.

In some embodiments, this data may be analyzed externally to the deviceand used to determine when the drug should be released: an externalsignal may then be sent to the device to trigger release of the drug.Thus, the communication system may further be adapted to receive asignal remotely triggering the actuator and thus causing release of theTLR agonist. The signal may be sent from an external transmitter inresponse to receipt/analysis and/or assessment of the environmentaldata, e.g., data indicating that the device has reached the desiredlocation of the gut (where the location of the diseased tissue has beenpre-determined) and/or data indicating the presence of diseased tissue.“External” may be “outside of the body”.

Thus, in another embodiment, the ingestible device may comprise:

an ingestible housing comprising a reservoir having a therapeuticallyeffective amount of the TLR agonist stored therein;

a release mechanism having a closed state which retains the TLR agonistin the reservoir and an open state which releases the TLR agonist fromthe reservoir to the exterior of the device;

an environmental detector for detecting environmental data indicatingthe location of the device in the gut and/or the presence of diseasedtissue;

a communication system for transmitting the environmental data to anexternal receiver and for receiving a signal from an externaltransmitter; and

an actuator which controls the transition of the release mechanism fromthe closed to the open state in response to the signal.

It will be understood from the above that when the device comprises oneor more environmental detectors, e.g., comprises an image detector, thecompositions may be used both for disease detection and for diseasetreatment.

Accordingly, in a further embodiment, there is provided a TLR agonistfor use in a method of detecting and treating a disease of thegastrointestinal tract in a subject, wherein the method comprises orallyadministering to the subject an ingestible device loaded with the TLRagonist, wherein the ingestible device comprises an environmental sensorfor determining the presence of diseased tissue in the GI tract, andwherein the TLR agonist is released by the device at a location in thegastrointestinal tract of the subject that is proximate to one or moresites of disease, as detected by the environmental sensor. The devicemay be according to any of the embodiments described herein.

In another embodiment, there is provided a composition for use in amethod of detecting and treating a disease of the gastrointestinal tractin a subject, wherein the composition comprises or consists of aningestible device loaded with a therapeutically effective amount of aTLR agonist, wherein the ingestible device comprises an environmentalsensor for determining the presence of diseased tissue in the GI tract,and wherein the TLR agonist is released by the device at a location inthe gastrointestinal tract of the subject that is proximate to one ormore sites of disease, as detected by the environmental sensor. Again,the device may be according to any of the embodiments described herein.

In some embodiments, where the ingestible device as used in the presentinvention comprises an environmental sensor for detecting the presenceof disease in the GI tract and a communication system as describedabove, the method of treatment may comprise:

i) receiving at an external receiver from the ingestible device a signaltransmitting the environmental data;

ii) assessing the environmental data to confirm the presence of thedisease; and

iii) when the presence of the disease is confirmed, sending from anexternal transmitter to the ingestible device a signal triggeringrelease of the TLR agonist.

For example, the presence of disease may be confirmed based on thepresence of inflamed tissue and/or lesions associated with any of thedisease states referred to herein. For example, the presence of diseasemay be confirmed based on the presence of inflammation, ulceration e.g.,aphthoid ulcerations, “punched-out ulcers” and/or superficial ulcers ofthe mucosa, cobblestoning, stenosis, granulomas, crypt abscesses,fissures, e.g., extensive linear fissures, villous atrophy, fibrosis,and/or bleeding.

In some embodiments, the present invention may relate to a systemcomprising:

an ingestible device loaded with a therapeutically effective amount of aTLR agonist, a release mechanism for release of the TLR agonist (e.g.,from a reservoir comprising the TLR agonist), an actuator controllingthe release mechanism, an environmental sensor for determining thelocation of the device in the gut and/or for detecting the presence ofdiseased tissue and a communication system adapted to transmit theenvironment data and receive a signal triggering the actuator;

a receiver and display module for receiving and displaying outside ofthe body the environment data from the ingestible device;

a transmitter for sending to the ingestible device a signal triggeringthe actuator.

In any of the above embodiments, the ingestible device may furthercomprise an anchoring system for anchoring the device or a portionthereof in a location and an actuator for the anchoring system. This maybe triggered in response to a determination that the device is at alocation in the gastrointestinal tract of the subject proximate to oneor more sites of disease. For instance, this may be detected by theenvironmental sensor. The triggering may be controlled by a processor inthe device, that is, autonomously. A device where the triggering iscontrolled by a processor in the device is said to be an autonomousdevice. Alternatively, it may be controlled by a signal sent fromoutside of the body, as described above.

In any of the above aspects and embodiments, disease of the GI tract maybe an inflammatory bowel disease.

In some embodiments, the disease of the GI tract is ulcerative colitis.

In some embodiments, the disease of the GI tract is Crohn's disease.

In general, apparatuses, compositions, and methods disclosed herein areuseful in the treatment of diseases of the gastrointestinal tract.Exemplary gastrointestinal tract diseases that can be treated include,without limitation, inflammatory bowel disease (IBD), Crohn's disease(e.g., active Crohn's disease, refractory Crohn's disease, orfistulizing Crohn's disease), ulcerative colitis, indeterminate colitis,microscopic colitis, infectious colitis, drug or chemical-inducedcolitis, diverticulitis, and ischemic colitis, gastritis, peptic ulcers,stress ulcers, bleeding ulcers, gastric hyperacidity, dyspepsia,gastroparesis, Zollinger-Ellison syndrome, gastroesophageal refluxdisease, short-bowel (anastomosis) syndrome, a hypersecretory stateassociated with systemic mastocytosis or basophilic leukemia orhyperhistaminemia, Celiac disease (e.g., nontropical Sprue), enteropathyassociated with seronegative arthropathies, microscopic colitis,collagenous colitis, eosinophilic gastroenteritis, colitis associatedwith radiotherapy or chemotherapy, colitis associated with disorders ofinnate immunity as in leukocyte adhesion deficiency-1, chronicgranulomatous disease, food allergies, gastritis, infectious gastritisor enterocolitis (e.g., Helicobacter pylori-infected chronic activegastritis), other forms of gastrointestinal inflammation caused by aninfectious agent, pseudomembranous colitis, hemorrhagic colitis,hemolytic-uremic syndrome colitis, diversion colitis, irritable bowelsyndrome, irritable colon syndrome, and pouchitis.

In some embodiments, apparatuses, compositions, and methods disclosedherein are used to treat one gastrointestinal disease. In someembodiments, apparatuses, compositions, and methods disclosed herein areused to treat more than one gastrointestinal disease. In someembodiments, apparatuses, compositions, and methods disclosed herein areused to treat multiple gastrointestinal diseases that occur in the samearea of the gastrointestinal tract (e.g., each disease can occur in thesmall intestine, large intestine, colon, or any sub-region thereof). Insome embodiments, apparatuses, compositions, and methods disclosedherein are used to treat multiple gastrointestinal diseases that occurin different areas of the gastrointestinal tract. In some embodiments,administration (e.g., local administration to the gastrointestinaltract) of TLR agonist is useful in the treatment of gastrointestinaldiseases including, but not limited to, inflammatory bowel disease(IBD), ulcerative colitis, Crohn's disease, or any of the othergastrointestinal diseases described herein.

Aspects and embodiments as described herein are intended to be freelycombinable. For example, any details or embodiments described herein formethods of treatment apply equally to a TLR agonist, composition oringestible device for use in said treatment. Any details or embodimentsdescribed for a device apply equally to methods of treatment using thedevice, or to a TLR agonist or composition for use in a method oftreatment involving the device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of an example embodiment of an ingestible device, inaccordance with some embodiments of the disclosure;

FIG. 2 is an exploded view of the ingestible device of FIG. 1, inaccordance with some embodiments of the disclosure;

FIG. 3 is a diagram of an ingestible device during an example transitthrough a GI tract, in accordance with some embodiments of thedisclosure;

FIG. 4 is a diagram of an ingestible device during an example transitthrough a jejunum, in accordance with some embodiments of thedisclosure;

FIG. 5 is a flowchart of illustrative steps for determining a locationof an ingestible device as it transits through a GI tract, in accordancewith some embodiments of the disclosure;

FIG. 6 is a flowchart of illustrative steps for detecting transitionsfrom a stomach to a duodenum and from a duodenum back to a stomach,which may be used when determining a location of an ingestible device asit transits through a GI tract, in accordance with some embodiments ofthe disclosure;

FIG. 7 is a plot illustrating data collected during an example operationof an ingestible device, which may be used when determining a locationof an ingestible device as it transits through a GI tract, in accordancewith some embodiments of the disclosure;

FIG. 8 is another plot illustrating data collected during an exampleoperation of an ingestible device, which may be used when determining alocation of an ingestible device as it transits through a GI tract, inaccordance with some embodiments of the disclosure;

FIG. 9 is a flowchart of illustrative steps for detecting a transitionfrom a duodenum to a jejunum, which may be used when determining alocation of an ingestible device as it transits through a GI tract, inaccordance with some embodiments of the disclosure;

FIG. 10 is a plot illustrating data collected during an exampleoperation of an ingestible device, which may be used when detecting atransition from a duodenum to a jejunum, in accordance with someembodiments of the disclosure;

FIG. 11 is a plot illustrating muscle contractions detected by aningestible device over time, which may be used when determining alocation of an ingestible device as it transits through a GI tract, inaccordance with some embodiments of the disclosure;

FIG. 12 is a flowchart of illustrative steps for detecting a transitionfrom a jejenum to an ileum, which may be used when determining alocation of an ingestible device as it transits through a GI tract, inaccordance with some embodiments of the disclosure;

FIG. 13 is a flowchart of illustrative steps for detecting a transitionfrom a jejenum to an ileum, which may be used when determining alocation of an ingestible device as it transits through a GI tract, inaccordance with some embodiments of the disclosure;

FIG. 14 is a flowchart of illustrative steps for detecting a transitionfrom an ileum to a cecum, which may be used when determining a locationof an ingestible device as it transits through a GI tract, in accordancewith some embodiments of the disclosure;

FIG. 15 is a flowchart of illustrative steps for detecting a transitionfrom a cecum to a colon, which may be used when determining a locationof an ingestible device as it transits through a GI tract, in accordancewith some embodiments of the disclosure;

FIG. 16 illustrates an ingestible device for delivering a substance inthe GI tract;

FIG. 17 illustrates aspects of a mechanism for an ingestible device witha gas generating cell configured to generate a gas to dispense asubstance;

FIG. 18 illustrates an ingestible device having a piston to push fordrug delivery;

FIG. 19 illustrates an ingestible device having a bellow structure for astorage reservoir of dispensable substances;

FIG. 20 illustrates an ingestible device having a flexible diaphragm todeform for drug delivery;

FIG. 21 shows an illustrative embodiment of an ingestible device withmultiple openings in the housing;

FIG. 22 shows a highly cross-section of an ingestible device including avalve system and a sampling system;

FIG. 23 illustrates a valve system;

FIGS. 24A and 24B illustrate a portion of a two-stage valve system inits first and second stages, respectively;

FIGS. 25A and 25B illustrate a portion of a two-stage valve system inits first and second stages, respectively;

FIGS. 26A and 26B illustrate a portion of a two-stage valve system inits first and second stages, respectively;

FIG. 27 illustrates a more detailed view of an ingestible deviceincluding a valve system and a sampling system;

FIG. 28 illustrates a portion of an ingestible device including asampling system and a two-stage valve system in its second stage; and

FIG. 29 is a highly schematic illustrate of an ingestible device.

FIG. 30 is a graph shiwng the percentage (%) change in body weight atday 14 (±SEM) for DSS mice treated with anti-IL-12 p40 antibodyintraperitoneally (10 mg/kg) every third day (Q3D) or intracecally (10mg/kg or 1 mg/kg) daily (QD), when compared to mice treated withanti-IL-12 p40 antibody intraperitoneally (10 mg/kg) every third day(Q3D) and vehicle control (Vehicle). Mann-Whitney's U¬-test andStudent's t-test were used for statistical analysis on non-Gaussian andGaussian data respectively. A value of p<0.05 was considered significant(Graph Pad Software, Inc.).

FIG. 31 is a graph showing the concentration of anti-IL-12 p40 rat IgG2A(μg/mL) in plasma of anti-IL-12 p40 intraperitoneally (10 mg/kg) andintracecally (10 mg/kg and 1 mg/kg) administered treatment groups givendaily (QD) or every third day (Q3D) when compared to vehicle control(Vehicle) and when IP is compared to IC. ELISA analysis was used todetermine the concentration of anti-IL-12 p40 (IgG2A). Data presented asmean±SEM. Mann-Whitney's U¬-test and Student's t-test were used forstatistical analysis on non-Gaussian and Gaussian data respectively. Avalue of p<0.05 was considered significant (Graph Pad Software, Inc.).

FIG. 32 is a graph showing the concentration of anti-IL-12 p40 antibody(IgG2A) (μg/mL) in the cecum and colon content of anti-IL-12 p40antibody intraperitoneally (10 mg/kg) and intracecally (10 mg/kg and 1mg/kg) administered treatment groups given daily (QD) or every third day(Q3D), when compared to vehicle control (Vehicle) and when IP iscompared to IC. ELISA analysis was used to determine the concentrationof rat IgG2A. Data presented as mean±SEM. Mann-Whitney's U-test andStudent's t-test were used for statistical analysis on non-Gaussian andGaussian data respectively. A value of p<0.05 was considered significant(Graph Pad Software, Inc.).

FIG. 33 is a graph showing the mean overall tissue immunolabel scores(intensity and extent) in acute DSS colitis mouse colon of anti-IL-12p40 antibody intracecally-treated versus vehicle control-treated DSSmice. Data presented as mean±SEM.

FIG. 34 is a graph showing the mean location-specific immunolabel scoresin acute DSS colitis mouse colon of anti-IL-12 p40 intracecally-treatedversus vehicle control-treated DSS mice. Data presented as mean±SEM.Mann-Whitney's U-test and Student's t-test were used for statisticalanalysis on non-Gaussian and Gaussian data respectively. A value ofp<0.05 was considered significant (Graph Pad Software, Inc.).

FIG. 35 is a graph showing the ratio of anti-IL-12 p40 antibody in thecolon tissue to the plasma concentration of the anti-IL-12 p40 antibodyin mice treated with the anti-IL-12 p40 antibody on day 0 (Q0) or day 3(Q3D) of the study, when measured at the same time point after theinitial dosing. An outlier animal was removed from Group 5.

FIG. 36 is a graph showing the concentration of Il-1β (μg/mL) in colontissue lysate of acute DSS colitis mice treated with anti-IL-12 p40intraperitoneally (10 mg/kg) every third day (Q3D) or intracecally (10mg/kg or 1 mg/kg) administered daily (QD), when compared to vehiclecontrol (Vehicle). Data presented as mean±SEM. Mann-Whitney's U-test andStudent's t-test were used for statistical analysis on non-Gaussian andGaussian data respectively. A value of p<0.05 was considered significant(Graph Pad Software, Inc.).

FIG. 37 is a graph showing the concentration of Il-6 (μg/mL) in colontissue lysate of acute DSS colitis mice treated with anti-IL-12 p40intraperitoneally (10 mg/kg) every third day (Q3D) or intracecally (10mg/kg or 1 mg/kg) administered daily (QD), when compared to vehiclecontrol (Vehicle). Data presented as mean±SEM. Mann-Whitney's U-test andStudent's t-test were used for statistical analysis on non-Gaussian andGaussian data respectively. A value of p<0.05 was considered significant(Graph Pad Software, Inc.

FIG. 38 is a graph showing the concentration of Il-17A (μg/mL) in colontissue lysate of acute DSS colitis mice treated with anti-IL-12 p40intraperitoneally (10 mg/kg) every third day (Q3D) or intracecally (10mg/kg and 1 mg/kg) administered daily (QD), when compared to vehiclecontrol (Vehicle). Data presented as mean±SEM. Mann-Whitney's U-test andStudent's t-test were used for statistical analysis on non-Gaussian andGaussian data respectively. A value of p<0.05 was considered significant(Graph Pad Software, Inc.).

FIG. 39 is a graph showing the percentage (%) change in body weight atday 14 (±SEM) for DSS mice treated with DATK32 (anti-α4β7) antibodyintraperitoneally (25 mg/kg) every third day (Q3D) or intracecally (25mg/kg or 5 mg/kg) administered daily (QD), when compared to vehiclecontrol (Vehicle) and when IC is compared to IP. Data presented asmean±SEM. Mann-Whitney's U-test and Student's t-test were used forstatistical analysis on non-Gaussian and Gaussian data respectively. Avalue of p<0.05 was considered significant (Graph Pad Software, Inc.).

FIG. 40 is a graph showing the plasma concentration of DATK32 rat IgG2A(μg/mL) of intraperitoneally (25 mg/kg) and intracecally (25 mg/kg and 5mg/kg) administered treatment groups given daily (QD) or every third day(Q3D), where IP is compared to IC. Data presented as mean±SEM.Mann-Whitney's U-test and Student's t-test were used for statisticalanalysis on non-Gaussian and Gaussian data respectively. A value ofp<0.05 was considered significant (Graph Pad Software, Inc.).

FIG. 41 is a graph showing the concentration of DATK32 rat IgG2Aantibody (μg/mL) in cecum and colon content of intraperitoneally (25mg/kg) or intracecally (25 mg/kg and 5 mg/kg) administered treatmentgroups given daily (QD) or every third day (Q3D), where IP is comparedto IC. Data presented as mean±SEM. Mann-Whitney's U-test and Student'st-test were used for statistical analysis on non-Gaussian and Gaussiandata respectively. A value of p<0.05 was considered significant (GraphPad Software, Inc.).

FIG. 42 is a graph showing the concentration of DATK32 rat IgG2A (μg/mL)in the colon content of intraperitoneally (25 mg/kg) or intracecally (25mg/kg and 5 mg/kg) administered treatment groups given daily (QD), andconcentration over time (1, 2, 4, 24, and 48 hours), where IP iscompared to IC. Data presented as mean±SEM. Mann-Whitney's U-test andStudent's t-test were used for statistical analysis on non-Gaussian andGaussian data respectively. A value of p<0.05 was considered significant(Graph Pad Software, Inc.).

FIG. 43 is a graph showing the concentration of DATK32 rat IgG2A (μg/g)in colon tissue of intraperitoneally (25 mg/kg) or intracecally (25mg/kg and 5 mg/kg) administered treatment groups given daily (QD) orevery third day (Q3D), where IP is compared to IC. Data presented asmean±SEM. Mann-Whitney's U-test and Student's t-test were used forstatistical analysis on non-Gaussian and Gaussian data respectively. Avalue of p<0.05 was considered significant (Graph Pad Software, Inc.).

FIG. 44 is a graph showing the concentration of DATK32 rat IgG2A (μg/g)in the colon tissue of intraperitoneally (25 mg/kg) or intracecally (25mg/kg and 5 mg/kg) administered treatment groups given daily (QD), andthe concentration over time (1, 2, 4, 24, and 48 hours) was determined,where IP is compared to IC. Data presented as mean±SEM. Mann-Whitney'sU-test and Student's t-test were used for statistical analysis onnon-Gaussian and Gaussian data respectively. A value of p<0.05 wasconsidered significant (Graph Pad Software, Inc.).

FIG. 45 is a graph showing the mean overall tissue immunolabel scores(intensity and extent) in acute DSS colitis mouse colon of DATK32(anti-α4β7) antibody treated versus vehicle control (Vehicle) treatedDSS mice. The data are presented as mean±SEM.

FIG. 46 is a graph showing the mean location-specific immunolabel scoresin acute DSS colitis mouse colon of DATK32 (anti-α4β7) antibody-treatedversus vehicle control (Vehicle)-treated DSS mice. Data presented asmean±SEM. Mann-Whitney's U-test and Student's t-test were used forstatistical analysis on non-Gaussian and Gaussian data respectively. Avalue of p<0.05 was considered significant (Graph Pad Software, Inc.).

FIG. 47 is a graph showing the ratio of the DATK-32 antibody in thecolon tissue to the plasma concentration of the DATK-32 antibody in micetreated with the DATK-32 antibody on day 0 (Q0) or day 3 (Q3D) of thestudy (Groups 9-12), when measured after initial dosing.

FIG. 48 is a graph showing the mean percentage of Th memory cells(mean±SEM) in blood for DATK32 (anti-α4β7) antibody intraperitoneally(25 mg/kg) or intracecally (25 mg/kg or 5 mg/kg) administered treatmentgroups given daily (QD) or every third day (Q3D), when compared tovehicle control (Vehicle) and when IP is compared to IC. Mean percentageTh memory cells were measured using FACS analysis. Data presented asmean±SEM. Mann-Whitney's U-test and Student's t-test were used forstatistical analysis on non-Gaussian and Gaussian data respectively. Avalue of p<0.05 was considered significant (Graph Pad Software, Inc.).

FIG. 49 is an exemplary image of a histological section of a distaltransverse colon of Animal 1501 showing no significant lesions (i.e.,normal colon).

FIG. 50 is an exemplary image of a histological section of a distaltransverse colon of Animal 2501 (treated with TNBS) showing areas ofnecrosis and inflammation.

FIG. 51 is a representative graph of plasma adalimumab concentrationsover time following a single subcutaneous (SQ) or topical administrationof adalimumab. The plasma concentrations of adalimumab were determined6, 12, 24, and 48 hours after administration of adalimumab. N/D=notdetectable.

FIG. 52 is a representative table of the plasma adalimumabconcentrations (μg/mL) as shown in FIG. 4.6.

FIG. 53 is a graph showing the concentration of TNFα (pg/mL per mg oftotal protein) in non-inflamed and inflamed colon tissue afterintracecal administration of adalimumab, as measured 6, 12, 24, and 24hours after the initial dosing.

FIG. 54 is a graph showing the concentration of TNFα (pg/mL per mg oftotal protein) in colon tissue after subcutaneous or intracecal(topical) administration of adalimumab, as measured 48 hours after theinitial dosing.

FIG. 55 is a graph showing the percentage (%) change in body weight atday 14 (±SEM) in acute DSS colitis mice treated with cyclosporine Aorally (10 mg/kg) every third day (Q3D) or intracecally (10 mg/kg or 3mg/kg) daily (QD), when compared to vehicle control (Vehicle). Datapresented as mean±SEM. Mann-Whitney's U-test and Student's t-test wereused for statistical analysis on non-Gaussian and Gaussian datarespectively. A value of p<0.05 was considered significant (Graph PadSoftware, Inc.).

FIG. 56 is a graph showing the plasma cyclosporine A (CsA) (ng/mL)concentration over time (1 h, 2 h, 4 h, and 24 h) in acute DSS colitismice treated daily (QD) with orally (PO) (10 mg/kg) or intracecally (IC)(10 mg/kg or 3 mg/kg) administered CsA. Data presented as mean±SEM.

FIG. 57 is a graph showing the colon tissue cyclosporine A (CsA) (ng/g)concentration over time (1 h, 2 h, 4 h and 24 h) in acute DSS colitismice treated daily (QD) with orally (PO) (10 mg/kg) or intracecally (IC)(10 mg/kg or 3 mg/kg) administered CsA. Data presented as mean±SEM.

FIG. 58 is a graph showing the peak colon tissue cyclosporine A (CsA)(ng/g) concentration in acute DSS colitis mice treated daily (QD) withorally (PO) (10 mg/kg) or intracecally (IC) (10 mg/kg or 3 mg/kg)administered CsA. Data presented as mean±SEM.

FIG. 59 is a graph showing the trough tissue concentration ofcyclosporine (CsA) (ng/g) in colon of acute DSS colitis mice treateddaily (QD) with orally (PO) (10 mg/kg) or intracecally (IC) (10 mg/kg or3 mg/kg) administered CsA. Data presented as mean±SEM.

FIG. 60 is a graph showing the interleukin-2 (Il-2) concentration(μg/mL) in colon tissue of acute DSS colitis mice treated daily (QD)with orally (PO) (10 mg/kg) or intracecally (IC) (10 mg/kg or 3 mg/kg)administered CsA, where PO is compared to IC. Data presented asmean±SEM. Mann-Whitney's U-test and Student's t-test were used forstatistical analysis on non-Gaussian and Gaussian data respectively. Avalue of p<0.05 was considered significant (Graph Pad Software, Inc.).

FIG. 61 is a graph showing the interleukin-6 (Il-6) concentration(μg/mL) in colon tissue of acute DSS colitis mice treated daily (QD)with orally (PO) (10 mg/kg) or intracecally (IC) (10 mg/kg or 3 mg/kg)administered CsA. Data presented as mean±SEM.

FIG. 62 illustrates a nonlimiting example of a system for collecting,communicating and/or analyzing data about a subject, using an ingestibledevice.

FIGS. 63A-F are graphs showing rat IgG2A concentration as measured in(A) colon homogenate, (B) mLN homogenate, (C) small intestinehomogenate, (D) cecum contents, (E) colon contents, and (F) plasma byELISA. Standards were prepared with plasma matrix. Samples were diluted1:50 before analysis. Sample 20 was removed from cecum contents analysisgraph (outlier). *p<0.05; **p<0.01; ****p<0.001 were determined usingthe unpaired t test.

FIG. 64 illustrates a tapered silicon bellows.

FIG. 65 illustrates a tapered silicone bellows in the simulated devicejig.

FIG. 66 illustrates a smooth PVC bellows.

FIG. 67 illustrates a smooth PVC bellows in the simulated device jig.

FIG. 68 demonstrates a principle of a competition assay performed in anexperiment.

FIG. 69 shows AlphaLISA data.

FIG. 70 shows AlphaLISA data.

FIG. 71 shows AlphaLISA data.

FIG. 72 is a flowchart of illustrative steps of a clinical protocol, inaccordance with some embodiments of the disclosure.

FIG. 73 is a graph showing the level of FAM-SMAD7-AS oligonucleotide inthe cecum tissue of DSS-induced colitis mice at 12-hours. The barsrepresent from left to right, Groups 2 through 5 in the experimentdescribed in Example 9.

FIG. 74 is a graph showing the level of FAM-SMAD7-AS oligonucleotide inthe colon tissue of DSS-induced colitis mice at 12-hours. The barsrepresent from left to right, Groups 2 through 5 in the experimentdescribed in Example 9.

FIG. 75 is a graph showing the level of FAM-SMAD7-AS oligonucleotide inthe cecum contents of DSS-induced colitis mice at 12-hours. The barsrepresent from left to right, Groups 2 through 5 in the experimentdescribed in Example 9.

FIG. 76 is a graph showing the mean concentration of tacrolimus in thececum tissue and the proximal colon tissue 12 hours after intra-cecal ororal administration of tacrolimus to swine as described in Example 10.

DETAILED DESCRIPTION

The present disclosure is directed to various methods and formulationsfor treating diseases of the gastrointestinal tract with an TLR agonist.For example, in an embodiment, a method of treating a disease of thegastrointestinal tract in a subject comprises administering to thesubject a pharmaceutical formulation comprising an TLR agonist whereinthe pharmaceutical formulation is released in the subject'sgastrointestinal tract proximate to one or more sites of disease. Forexample, in an embodiment, the pharmaceutical formulation comprises atherapeutically effective amount of an TLR agonist.

In some embodiments, the formulation is contained in an ingestibledevice, and the device releases the formulation at a location proximateto the site of disease. The location of the site of disease may bepredetermined. For example, an ingestible device, the location of whichwithin the GI tract can be accurately determined as disclosed herein,may be used to sample one or more locations in the GI tract and todetect one or more analytes, including markers of the disease, in the GItract of the subject. A pharmaceutical formulation may be thenadministered via an ingestible device and released at a locationproximate to the predetermined site of disease. The release of theformulation may be triggered autonomously, as further described herein.

The following disclosure illustrates aspects of the formulations andmethods embodied in the claims.

Formulations, Including Pharmaceutical Formulations

As used herein, a “formulation” of an TLR agonist may refer to eitherthe TLR agonist in pure form, such as, for example, a lyophilized TLRagonist, or a mixture of the TLR agonist with one or morephysiologically acceptable carriers, excipients or stabilizers. Thus,therapeutic formulations or medicaments can be prepared by mixing theTLR agonist having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) antibody; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn— protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Exemplary pharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX<®>, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.Exemplary lyophilized formulations are described in U.S. Pat. No.6,267,958. Aqueous formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

A formulation of an TLR agonist as disclosed herein, e.g.,sustained-release formulations, can further include a mucoadhesiveagent, e.g., one or more of polyvinyl pyrolidine, methyl cellulose,sodium carboxyl methyl cellulose, hydroxyl propyl cellulose, carbopol, apolyacrylate, chitosan, a eudragit analogue, a polymer, and a thiomer.Additional examples of mucoadhesive agents that can be included in aformulation with an TLR agonist are described in, e.g., Peppas et al.,Biomaterials 17(16):1553-1561, 1996; Kharenko et al., PharmaceuticalChemistry J. 43(4):200-208, 2009; Salamat-Miller et al., Adv. DrugDeliv. Reviews 57(11):1666-1691, 2005; Bernkop-Schnurch, Adv. DrugDeliv. Rev. 57(11):1569-1582, 2005; and Harding et al., Biotechnol.Genet. Eng. News 16(1):41-86, 1999.

In some embodiments, components of a formulation may include any one ofthe following components, or any combination thereof:

Acacia, Alginate, Alginic Acid, Aluminum Acetate, an antiseptic, BenzylAlcohol, Butyl Paraben, Butylated Hydroxy Toluene, an antioxidant.Citric acid, Calcium carbonate, Candelilla wax, a binder, Croscarmellosesodium, Confectioner sugar, Colloidal silicone dioxide, Cellulose,Carnuba wax, Corn starch, Carboxymethylcellulose calcium, Calciumstearate, Calcium disodium EDTA, Chelation agents, Copolyvidone, Castoroil hydrogenated, Calcium hydrogen phosphate dehydrate, Cetylpyridinechloride, Cysteine HCl, Crosspovidone, Dibasic Calcium Phosphate,Disodium hydrogen phosphate, Dimethicone, Erythrosine Sodium, EthylCellulose, Gelatin, Glyceryl monooleate, Glycerin, Glycine, Glycerylmonostearate, Glyceryl behenate, Hydroxy propyl cellulose, Hydroxylpropyl methyl cellulose, Hypromellose, HPMC Pthalate, Iron oxides orferric oxide, Iron oxide yellow, Iron oxide red or ferric oxide, Lactose(hydrous or anhydrous or monohydrate or spray dried), Magnesiumstearate, Microcrystalline cellulose, Mannitol, Methyl cellulose,Magnesium carbonate, Mineral oil, Methacrylic acid copolymer, Magnesiumoxide, Methyl paraben, PEG, Polysorbate 80, Propylene glycol,Polyethylene oxide, Propylene paraben, Polaxamer 407 or 188 or plain,Potassium bicarbonate, Potassium sorbate, Potato starch, Phosphoricacid, Polyoxy 140 stearate, Sodium starch glycolate, Starchpregelatinized, Sodium crossmellose, Sodium lauryl sulfate, Starch,Silicon dioxide, Sodium benzoate, Stearic acid, Sucrose base formedicated confectionery, a granulating agent, Sorbic acid, Sodiumcarbonate, Saccharin sodium, Sodium alginate, Silica gel, Sorbitonmonooleate, Sodium stearyl fumarate, Sodium chloride, Sodiummetabisulfite, Sodium citrate dehydrate, Sodium starch, Sodium carboxymethyl cellulose, Succinic acid, Sodium propionate, Titanium dioxide,Talc, Triacetin, Triethyl citrate.

Accordingly, in some embodiments of the method of treating a disease asdisclosed herein, the method comprises administering to the subject apharmaceutical composition that is a formulation as disclosed herein. Insome embodiments the formulation is a dosage form, which may be, as anexample, a solid form such as, for example, a capsule, a tablet, asachet, or a lozenge; or which may be, as an example, a liquid form suchas, for example, a solution, a suspension, an emulsion, or a syrup.

In some embodiments, the formulation is not comprised in an ingestibledevice. In some embodiments wherein the formulation is not comprised inan ingestible device, the formulation may be suitable for oraladministration. The formulation may be, for example, a solid dosage formor a liquid dosage form as disclosed herein. In some embodiments whereinthe formulation is not comprised in an ingestible device, theformulation may be suitable for rectal administration. The formulationmay be, for example, a dosage form such as a suppository or an enema. Inembodiments where the formulation is not comprised in an ingestibledevice, the formulation releases the TLR agonist at a location in thegastrointestinal tract of the subject that is proximate to one or moresites of disease. Such localized release may be achieved, for example,with a formulation comprising an enteric coating. Such localized releasemay be achieved, an another example, with a formulation comprising acore comprising one or more polymers suitable for controlled release ofan active substance. A non-limiting list of such polymers includes:poly(2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethylmethacrylate, poly(ethylene glycol), poly(2-aminoethyl methacrylate),(2-hydroxypropyl)methacrylamide, poly(β-benzyl-1-aspartate),poly(N-isopropylacrylamide), and cellulose derivatives.

In some embodiments, the formulation is comprised in an ingestibledevice as disclosed herein. In some embodiments wherein the formulationis comprised in an ingestible device, the formulation may be suitablefor oral administration. The formulation may be, for example, a soliddosage form or a liquid dosage form as disclosed herein. In someembodiments the formulation is suitable for introduction and optionallyfor storage in the device. In some embodiments the formulation issuitable for introduction and optionally for storage in a reservoircomprised in the device. In some embodiments the formulation is suitablefor introduction and optionally for storage in a reservoir comprised inthe device. Thus, in some embodiments, provided herein is a reservoircomprising a therapeutically effective amount of an TLR agonist, whereinthe reservoir is configured to fit into an ingestible device. In someembodiments, the reservoir comprising a therapeutically effective amountof an TLR agonist is attachable to an ingestible device. In someembodiments, the reservoir comprising a therapeutically effective amountof an TLR agonist is capable of anchoring itself to the subject'stissue. As an example, the reservoir capable of anchoring itself to thesubject's tissue comprises silicone. As an example, the reservoircapable of anchoring itself to the subject's tissue comprises polyvinylchloride.

In some embodiments the formulation is suitable for introduction in aspray catheter, as disclosed herein.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, for example,those with complementary activities that do not adversely affect eachother. For instance, the formulation may further comprise another TLRagonist or a chemotherapeutic agent. Such molecules are suitably presentin combination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the TLR agonist, which matrices are inthe form of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated TLR agonists remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Pharmaceutical formulations may contain one or more TLR agonists. Thepharmaceutical formulations may be formulated in any manner known in theart. In some embodiments the formulations include one or more of thefollowing components: a sterile diluent (e.g., sterile water or saline),a fixed oil, polyethylene glycol, glycerin, propylene glycol, or othersynthetic solvents, antibacterial or antifungal agents, such as benzylalcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like, antioxidants, such as ascorbic acid or sodiumbisulfite, chelating agents, such as ethylenediaminetetraacetic acid,buffers, such as acetates, citrates, or phosphates, and isotonic agents,such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol orsorbitol), or salts (e.g., sodium chloride), or any combination thereof.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers (see, e.g., U.S. Pat. No. 4,522,811, incorporated by referenceherein in its entirety). The formulations can be formulated and enclosedin ampules, disposable syringes, or multiple dose vials. Where required,proper fluidity can be maintained by, for example, the use of a coating,such as lecithin, or a surfactant. Controlled release of the TLR agonistcan be achieved by implants and microencapsulated delivery systems,which can include biodegradable, biocompatible polymers (e.g., ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid; Alza Corporation and NovaPharmaceutical, Inc.).

In some embodiments, the TLR agonist is present in a pharmaceuticalformulation within the device.

In some embodiments, the TLR agonist is present in solution within thedevice.

In some embodiments, the TLR agonist is present in a suspension in aliquid medium within the device.

In some embodiments, the TLR agonist is present as a pure, powder (e.g.,lyophilized) form of the TLR agonist.

Definitions

By “ingestible”, it is meant that the device can be swallowed whole.

“Gastrointestinal inflammatory disorders” are a group of chronicdisorders that cause inflammation and/or ulceration in the mucousmembrane. These disorders include, for example, inflammatory boweldisease (e.g., Crohn's disease, ulcerative colitis, indeterminatecolitis and infectious colitis), mucositis (e.g., oral mucositis,gastrointestinal mucositis, nasal mucositis and proctitis), necrotizingenterocolitis and esophagitis.

“Inflammatory Bowel Disease” or “IBD” is a chronic inflammatoryautoimmune condition of the gastrointestinal (GI) tract. The GI tractcan be divided into four main different sections, the oesophagus,stomach, small intestine and large intestine or colon. The smallintestine possesses three main subcompartments: the duodenum, jejunumand ileum. Similarly, the large intestine consists of six sections: thececum, ascending colon, transverse colon, ascending colon, sigmoidcolon, and the rectum. The small intestine is about 6 m long, itsdiameter is 2.5 to 3 cm and the transit time through it is typically 3hours. The duodenum has a C-shape, and is 30 cm long. Due to its directconnection with the stomach, it is physically more stable than thejejunum and ileum, which are sections that can freely move. The jejunumis 2.4 m in length and the ileum is 3.6 m in length and their surfaceareas are 180 m² and 280 m² respectively. The large intestine is 1.5 mlong, its diameter is between 6.3 and 6.5 cm, the transit time thoughthis section is 20 hours and has a reduced surface area of approximately150 m². The higher surface area of the small intestine enhances itscapacity for systemic drug absorption.

The etiology of IBD is complex, and many aspects of the pathogenesisremain unclear. The treatment of moderate to severe IBD posessignificant challenges to treating physicians, because conventionaltherapy with corticosteroids and immunomodulator therapy (e.g.,azathioprine, 6 mercaptopurine, and methotrexate administered viatraditional routes such as tablet form, oral suspension, orintravenously) is associated with side effects and intolerance and hasnot shown proven benefit in maintenance therapy (steroids). Monoclonalantibodies targeting tumor necrosis factor alpha (TNF-α), such asinfliximab (a chimeric antibody) and adalimumab (a fully humanantibody), are currently used in the management of CD. Infliximab hasalso shown efficacy and has been approved for use in UC. However,approximately 10%-20% of patients with CD are primary nonresponders toanti TNF therapy, and another ˜20%-30% of CD patients lose response overtime (Schnitzler et al., Gut 58:492-500 (2009)). Other adverse events(AEs) associated with anti TNFs include elevated rates of bacterialinfection, including tuberculosis, and, more rarely, lymphoma anddemyelination (Chang et al., Nat Clin Pract Gastroenterol Hepatology3:220 (2006); Hoentjen et al., World J. Gastroenterol. 15(17):2067(2009)). No currently available therapy achieves sustained remission inmore than 20%-30% of IBD patients with chronic disease (Hanauer et al,Lancet 359: 1541-49 (2002); Sandborn et al, N Engl J Med 353: 1912-25(2005)). In addition, most patients do not achieve sustainedsteroid-free remission and mucosal healing, clinical outcomes thatcorrelate with true disease modification.

Although the cause of IBD remains unknown, several factors such asgenetic, infectious and immunologic susceptibility have been implicated.IBD is much more common in Caucasians, especially those of Jewishdescent. The chronic inflammatory nature of the condition has promptedan intense search for a possible infectious cause. Although agents havebeen found which stimulate acute inflammation, none has been found tocause the chronic inflammation associated with IBD. The hypothesis thatIBD is an autoimmune disease is supported by the previously mentionedextraintestinal manifestation of IBD as joint arthritis, and the knownpositive response to IBD by treatment with therapeutic agents such asadrenal glucocorticoids, cyclosporine and azathioprine, which are knownto suppress immune response. In addition, the GI tract, more than anyother organ of the body, is continuously exposed to potential antigenicsubstances such as proteins from food, bacterial byproducts (LPS), etc.

A chronic inflammatory autoimmune condition of the gastrointestinal (GI)tract presents clinically as either ulcerative colitis (UC) or Crohn'sdisease (CD). Both IBD conditions are associated with an increased riskfor malignancy of the GI tract.

“Crohn's disease” (“CD”) is a chronic transmural inflammatory diseasewith the potential to affect any part of the entire GI tract, and UC isa mucosal inflammation of the colon. Both conditions are characterizedclinically by frequent bowel motions, malnutrition, and dehydration,with disruption in the activities of daily living.

CD is frequently complicated by the development of malabsorption,strictures, and fistulae and may require repeated surgery. UC, lessfrequently, may be complicated by severe bloody diarrhea and toxicmegacolon, also requiring surgery. The most prominent feature Crohn'sdisease is the granular, reddish-purple edematous thickening of thebowel wall. With the development of inflammation, these granulomas oftenlose their circumscribed borders and integrate with the surroundingtissue. Diarrhea and obstruction of the bowel are the predominantclinical features. As with ulcerative colitis, the course of Crohn'sdisease may be continuous or relapsing, mild or severe, but unlikeulcerative colitis, Crohn's disease is not curable by resection of theinvolved segment of bowel. Most patients with Crohn's disease requiresurgery at some point, but subsequent relapse is common and continuousmedical treatment is usual. Crohn's disease may involve any part of thealimentary tract from the mouth to the anus, although typically itappears in the ileocolic, small-intestinal or colonic-anorectal regions.Histopathologically, the disease manifests by discontinuousgranulomatomas, crypt abscesses, fissures and aphthous ulcers. Theinflammatory infiltrate is mixed, consisting of lymphocytes (both T andB cells), plasma cells, macrophages, and neutrophils. There is adisproportionate increase in IgM- and IgG-secreting plasma cells,macrophages and neutrophils.

To date, the primary outcome measure in Crohn's Disease clinical trialsis the Crohn's Disease Activity Index (CDAI), which has served as thebasis for approval of multiple drug treatments, including for example,vedolizumab and natalizumab. The CDAI was developed by regressingclinician global assessment of disease activity on eighteen potentialitems representing patient reported outcomes (PROs) (i.e. abdominalpain, pain awakening patient from sleep, appetite), physical signs (i.e.average daily temperature, abdominal mass), medication use (i.e.loperamide or opiate use for diarrhea) and a laboratory test (i.e.hematocrit). Backward stepwise regression analysis identified eightindependent predictors which are the number of liquid or soft stools,severity of abdominal pain, general well-being, occurrence ofextra-intestinal symptoms, need for anti-diarrheal drugs, presence of anabdominal mass, hematocrit, and body weight. The final score is acomposite of these eight items, adjusted using regression coefficientsand standardization to construct an overall CDAI score, ranging from 0to 600 with higher score indicating greater disease activity. Widelyused benchmarks are: CDAI <150 is defined as clinical remission, 150 to219 is defined as mildly active disease, 220 to 450 is defined asmoderately active disease, and above 450 is defined as very severedisease (Best W R, et al., Gastroenterology 77:843-6, 1979). Vedolizumaband natalizumab have been approved on the basis of demonstrated clinicalremission, i.e. CDAI <150.

Although the CDAI has been in use for over 40 years, and has served asthe basis for drug approval, it has several limitations as an outcomemeasure for clinical trials. For example, most of the overall scorecomes from the patient diary card items (pain, number of liquid bowelmovements, and general well-being), which are vaguely defined and notstandardized terms (Sandler et al., J. Clin. Epidemiol 41:451-8, 1988;Thia et al., Inflamm Bowel Dis 17: 105-11, 2011). In addition,measurement of pain is based on a four-point scale rather than anupdated seven-point scale. The remaining 5 index items contribute verylittle to identifying an efficacy signal and may be a source ofmeasurement noise. Furthermore, concerns have been raised about poorcriterion validity for the CDAI, a reported lack of correlation betweenthe CDAI and endoscopic measures of inflammation (which may render theCDAI as a poor discriminator of active CD and irritable bowel syndrome)and high reported placebo rates (Korzenik et al., N Engl J Med.352:2193-201, 2005; Sandborn W J, et al., N Engl J Med 353: 1912-25,2005; Sandborn W J, et al., Ann Intern 19; 146:829-38, 2007, Epub 2007Apr. 30; Kim et al., Gastroenterology 146: (5 supplement 1) S-368,2014).

It is, thus, generally recognized that additional or alternativemeasures of CD symptoms are needed, such as new PRO tools or adaptationsof the CDAI to derive a new PRO. The PRO2 and PRO3 tools are suchadaptations of the CDAI and have been recently described in Khanna etal., Aliment Pharmacol. Ther. 41: 77-86, 2015. The PRO2 evaluates thefrequency of loose/liquid stools and abdominal pain {Id). These itemsare derived and weighted accordingly from the CDAI and are the CDAIdiary card items, along with general well-being, that contribute most tothe observed clinical benefit measured by CDAI (Sandler et al., J. Clin.Epidemiol 41:451-8, 1988; Thia et al., Inflamm Bowel Dis 17: 105-11,2011; Kim et al., Gastroenterology 146: (5 supplement 1) S-368, 2014).The remission score of <11 is the CDAI-weighted sum of the average stoolfrequency and pain scores in a 7-day period, which yielded optimumsensitivity and specificity for identification of CDAI remission (scoreof <150) in a retrospective data analysis of ustekinumab inductiontreatment for moderate to severe CD in a Phase II clinical study (GasinkC, et al., abstract, ACG Annual Meeting 2014). The PRO2 was shown to besensitive and responsive when used as a continuous outcome measure in aretrospective data analysis of MTX treatment in active CD (Khanna R, etal., Inflamm Bowel Dis 20: 1850-61, 2014) measured by CDAI. Additionaloutcome measures include the Mayo Clinic Score, the Crohn diseaseendoscopic index of severity (CDEIS), and the Ulcerative colitisendoscopic index of severity (UCEIS). Additional outcome measuresinclude Clinical remission, Mucosal healing, Histological healing(transmural), MRI or ultrasound for measurement or evaluation of bowelwall thickness, abscesses, fistula and histology.

An additional means of assessing the extent and severity of Crohn'sDisease is endoscopy. Endoscopic lesions typical of Crohn's disease havebeen described in numerous studies and include, e.g., aphthoidulcerations, “punched-out ulcers,” cobblestoning and stenosis.Endoscopic evaluation of such lesions was used to develop the firstvalidated endoscopic score, the Crohn's Disease Endoscopic Index ofSeverity (CDEIS) (Mary et al., Gut 39:983-9, 1989). More recently,because the CDEIS is time-consuming, complicated and impractical forroutine use, a Simplified Endoscopic Activity Score for Crohn's Disease(SES-CD) was developed and validated (Daperno et al., Gastrointest.Endosc. 60(4):505-12, 2004). The SES-CD consists of four endoscopicvariables (size of ulcers, proportion of surface covered by ulcers,proportion of surface with any other lesions (e.g., inflammation), andpresence of narrowings [stenosis]) that are scored in five ileocolonicsegments, with each variable, or assessment, rated from 0 to 3.

To date, there is no cure for CD. Accordingly, the current treatmentgoals for CD are to induce and maintain symptom improvement, inducemucosal healing, avoid surgery, and improve quality of life(Lichtenstein G R, et al., Am J Gastroenterol 104:465-83, 2009; VanAssche G, et al., J Crohns Colitis. 4:63-101, 2010). The current therapyof IBD usually involves the administration of antiinflammatory orimmunosuppressive agents, such as sulfasalazine, corticosteroids,6-mercaptopurine/azathioprine, or cyclosporine, all of which are nottypically delivered by localized release of a drug at the site orlocation of disease. More recently, biologics like TNF-alpha inhibitorsand IL-12/IL-23 blockers, are used to treat IBD. Ifanti-inflammatory/immunosuppressive/biologic therapies fail, colectomiesare the last line of defense. The typical operation for CD not involvingthe rectum is resection (removal of a diseased segment of bowel) andanastomosis (reconnection) without an ostomy. Sections of the small orlarge intestine may be removed. About 30% of CD patients will needsurgery within the first year after diagnosis. In the subsequent years,the rate is about 5% per year. Unfortunately, CD is characterized by ahigh rate of recurrence; about 5% of patients need a second surgery eachyear after initial surgery.

Refining a diagnosis of inflammatory bowel disease involves evaluatingthe progression status of the diseases using standard classificationcriteria. The classification systems used in IBD include the Trueloveand Witts Index (Truelove S. C. and Witts, L. J. Br Med J. 1955; 2:1041-1048), which classifies colitis as mild, moderate, or severe, aswell as Lennard-Jones. (Lennard-Jones J E. Scand J Gastroenterol Suppl1989; 170:2-6) and the simple clinical colitis activity index (SCCAI).(Walmsley et. al. Gut. 1998; 43:29-32) These systems track suchvariables as daily bowel movements, rectal bleeding, temperature, heartrate, hemoglobin levels, erythrocyte sedimentation rate, weight,hematocrit score, and the level of serum albumin.

There is sufficient overlap in the diagnostic criteria for UC and CDthat it is sometimes impossible to say which a given patient has;however, the type of lesion typically seen is different, as is thelocalization. UC mostly appears in the colon, proximal to the rectum,and the characteristic lesion is a superficial ulcer of the mucosa; CDcan appear anywhere in the bowel, with occasional involvement ofstomach, esophagus and duodenum, and the lesions are usually describedas extensive linear fissures.

In approximately 10-15% of cases, a definitive diagnosis of ulcerativecolitis or Crohn's disease cannot be made and such cases are oftenreferred to as “indeterminate colitis.” Two antibody detection tests areavailable that can help the diagnosis, each of which assays forantibodies in the blood. The antibodies are “perinuclear anti-neutrophilantibody” (pANCA) and “anti-Saccharomyces cervisiae antibody” (ASCA).Most patients with ulcerative colitis have the pANCA antibody but notthe ASCA antibody, while most patients with Crohn's disease have theASCA antibody but not the pANCA antibody. However, these two tests haveshortcomings as some patients have neither antibody and some Crohn'sdisease patients may have only the pANCA antibody. A third test, whichmeasures the presence and accumulation of circulating anti-microbialantibodies—particularly flagellin antibodies, has proven to be usefulfor detecting susceptibility to Crohn's Disease before diseasedevelopment. See Choung, R. S., et al. “Serologic microbial associatedmarkers can predict Crohn's disease behaviour years before diseasediagnosis.” Alimentary pharmacology & therapeutics 43.12 (2016):1300-1310.

“Ulcerative colitis (UC)” afflicts the large intestine. The course ofthe disease may be continuous or relapsing, mild or severe. The earliestlesion is an inflammatory infiltration with abscess formation at thebase of the crypts of Lieberkuhn. Coalescence of these distended andruptured crypts tends to separate the overlying mucosa from its bloodsupply, leading to ulceration. Symptoms of the disease include cramping,lower abdominal pain, rectal bleeding, and frequent, loose dischargesconsisting mainly of blood, pus and mucus with scanty fecal particles. Atotal colectomy may be required for acute, severe or chronic,unremitting ulcerative colitis.

The clinical features of UC are highly variable, and the onset may beinsidious or abrupt, and may include diarrhea, tenesmus and relapsingrectal bleeding. With fulminant involvement of the entire colon, toxicmegacolon, a life-threatening emergency, may occur. Extraintestinalmanifestations include arthritis, pyoderma gangrenoum, uveitis, anderythema nodosum.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for example, fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecific,trispecific etc. antibodies so long as they exhibit the desiredbiological activity) and may also include certain antibody fragments (asdescribed in greater detail herein). An antibody can be human, humanizedand/or affinity matured.

“Antibody fragments” comprise only a portion of an intact antibody,where in certain embodiments, the portion retains at least one, andtypically most or all, of the functions normally associated with thatportion when present in an intact antibody. In one embodiment, anantibody fragment comprises an antigen binding site of the intactantibody and thus retains the ability to bind antigen. In anotherembodiment, an antibody fragment, for example one that comprises the Fcregion, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody, suchas FcRn binding, antibody half-life modulation, ADCC function andcomplement binding. In one embodiment, an antibody fragment is amonovalent antibody that has an in vivo half-life substantially similarto an intact antibody. For example, such an antibody fragment maycomprise on antigen binding arm linked to an Fc sequence capable ofconferring in vivo stability to the fragment.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Treatment regimen” refers to a combination of dosage, frequency ofadministration, or duration of treatment, with or without addition of asecond medication.

“Effective treatment regimen” refers to a treatment regimen that willoffer beneficial response to a patient receiving the treatment.

“Effective amount” refers to an amount of drug that offers beneficialresponse to a patient receiving the treatment. For example, an effectiveamount may be a Human Equivalent Dose (HED).

“Dispensable”, with reference to any substance, refers to any substancethat may be released from an ingestible device as disclosed herein, orfrom a component of the device such as a reservoir. For example, adispensable substance may be an TLR agonist, and/or a formulationcomprising an TLR agonist.

“Patient response” or “patient responsiveness” can be assessed using anyendpoint indicating a benefit to the patient, including, withoutlimitation, (1) inhibition, to some extent, of disease progression,including slowing down and complete arrest; (2) reduction in the numberof disease episodes and/or symptoms; (3) reduction in lesional size; (4)inhibition (i.e., reduction, slowing down or complete stopping) ofdisease cell infiltration into adjacent peripheral organs and/ortissues; (5) inhibition (i.e., reduction, slowing down or completestopping) of disease spread; (6) decrease of auto-immune response, whichmay, but does not have to, result in the regression or ablation of thedisease lesion; (7) relief, to some extent, of one or more symptomsassociated with the disorder; (8) increase in the length of disease-freepresentation following treatment; and/or (9) decreased mortality at agiven point of time following treatment. The term “responsiveness”refers to a measurable response, including complete response (CR) andpartial response (PR).

As used herein, “complete response” or “CR” means the disappearance ofall signs of inflammation or remission in response to treatment. Thisdoes not necessarily mean the disease has been cured.

“Partial response” or “PR” refers to a decrease of at least 50% in theseverity of inflammation, in response to treatment.

A “beneficial response” of a patient to treatment with a therapeuticagent and similar wording refers to the clinical or therapeutic benefitimparted to a patient at risk for or suffering from a gastrointestinalinflammatory disorder from or as a result of the treatment with theagent. Such benefit includes cellular or biological responses, acomplete response, a partial response, a stable disease (withoutprogression or relapse), or a response with a later relapse of thepatient from or as a result of the treatment with the agent.

As used herein, “non-response” or “lack of response” or similar wordingmeans an absence of a complete response, a partial response, or abeneficial response to treatment with a therapeutic agent.

“A patient maintains responsiveness to a treatment” when the patient'sresponsiveness does not decrease with time during the course of atreatment.

A “symptom” of a disease or disorder (e.g., inflammatory bowel disease,e.g., ulcerative colitis or Crohn's disease) is any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by a subject and indicative of disease.

TLR Modulators

A “TLR modulator” is an agent that functionally interacts with atoll-like receptor (TLR) expressed in a mammalian cell (e.g., a humancell). In some embodiments, the modulator is a TLR agonist. In someembodiments, the modulator is a TLR antagonist.

TLR Agonists

The term “TLR agonist” is an agent that binds to and activates atoll-like receptor (TLR) expressed in a mammalian cell (e.g., a humancell). In some embodiments, the TLR agonist binds to and activates TLR1.In some embodiments, the TLR agonist binds to and activates TLR2. Insome embodiments, the TLR agonist binds to and activates TLR3. In someembodiments, the TLR agonist binds to and activates TLR4. In someembodiments, the TLR agonist binds to and activates TLR5. In someembodiments, the TLR agonist binds to and activates TLR6. In someembodiments, the TLR agonist binds to and activates TLR7. In someembodiments, the TLR agonist binds to and activates TLR8. In someembodiments, the TLR agonist binds to and activates TLR9. In someembodiments, the TLR agonist binds to and activates TLR10. In someembodiments, the TLR agonist binds to and activates TLR11. In someembodiments, the TLR agonist binds to and activates two or more (e.g.,three, four, five, six, seven, eight, nine, ten, or eleven) TLRs (e.g.,two or more of any of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, and TLR11 (in any combination)).

In some embodiments, the TLR agonist is a synthetic TLR agonist, a TLRmimic, or a small molecule. Non-limiting examples of TLR agonists aredescribed in Bhardwaj et al., Cancer J. 16(4):382-391, 2010; Meyer etal., Exp. Opin. Investig. Drugs 17(7):1051-1065, 2008; Adams,Immunotherapy 1(6):949-964, 2009; Hennessy et al., Nat. Rev. DrugDiscov. 9:293-307, 2010; and U.S. Pat. Nos. 7,498,409; 9,421,254;8,409,813; 8,361,986; 8,795,678; 8,728,486; 8,636,979; 8,999,946;9,359,360; 9,050,376; and 9,556,167; US 2014/0322271; US 2016/0206690;US 2009/0253622; US 2011/0135669; US 2011/0250175; US 2014/0220074; andUS 2012/0219615; each incorporated in its entirety herein. In someembodiments, the TLR agonist is a peptide or a fusion protein (Huleattet al., Vaccine 25: 763-775, 2007).

In some embodiments, a TLR agonist specifically binds to and activates asingle TLR (e.g., TLR4, TLR7, TLR8, or TLR9; Zhu et al., J. Clin.Invest. 120:607-616, 2010; Zhu et al., PNAS 105:16260-16265, 2008; Wanget al., J. Virol. 79(22):14355-14370, 2005). In some embodiments, theTLR agonist binds to and activates more than one TLR (e.g., Bacillus ofCalmette-Guerin, Myobacterium bovis (BCG); Morton et al., Ann. Surg.180(4):635-643, 1974; Mortoon et al., J. Clin. Oncol. ASCO Ann. MeetingProceedings Part I 25(18 Suppl), 2007). In some embodiments, the TLRagonist is a TLR2/TLR6 agonist (e.g., Pam₂CSK4 or MALP-2 (Agnihotri etal., J. Med. Chem. 54: 8148-8160, 2011; Wu et al., J. Med. Chem. 53:3198-3213, 2010)).

In some embodiments, the TLR agonist is administrated in combinationwith another composition (Dowling et al., Clin. Transl. Immunol. 5:e85,2016). In some embodiments, the TLR agonist is an endogenous moleculereleased from dead cells (e.g., a heat shock protein (HSP) and mobilitygroup box 1 (HMGB1); Asea et al., J. Biol. Chem. 277:15028-15034, 2002;Kepp et al., Cancer Metastasis 30: 61-69, 2011).

TLR3 Agonists

In some embodiments, the TLR agonist specifically binds and activatesTLR3 (e.g., a synthetic agonist). Non-limiting examples of TLR agoniststhat bind and activate TLR3 are described in Nicodemus et al.,Immunotherapy 2:137-140, 2010. In some embodiments, the TLR3 agonist isa synthetic double-stranded RNA (dsRNA) complex (e.g., polyribosinic:polyribocytidic acid (polyI:C); Sivori et al., PNAS 101:10116-10121,2004; Sloat et al., Pharmaceutical Res. 23:1217-1226, 2006; Ichinohe etal., Microbes and infection/Institut Pasteur 9:1333-1340, 2007; Robinsonet al., J. Natl. Cancer Inst. 57(3):599-602, 1976). In some embodiments,the TLR3 agonist is a TLR3 mimic (e.g., polyadenosine-polyuridylic acid(poly A:U) (Veyrat et al., Oncotarget 7(50):82580-82593, 2016; Alizadehet al., Iran J. Allergy Asthma Immunol. 12(2):161-167, 2013);rintatolimod (polyI: polyCU, Ampligen®) (Steinman et al., Nature 449:419-426, 2007; Jasani et al., Vaccine 27(25-26):3401-3404, 2009; Strayeret al., PLoS One 7(3): e31334, 2012). In some embodiments, the TLR3mimic is polyionisinic-polycytidylic acid stabilized with poly-L-lysineand carboxymethylcellulose (Poly-ICLC, Hiltonol®; Hawkins et al., J.Biol. Resp. Mod. 4:664-668, 1985; Butowski et al., J. Neurooncol.91:175-182, 2009; Jeong et al., J. Neurochem. doi.10.1111, 2015). Insome embodiments, the TLR3 agonist is RGC100 (Naumann et al., Clin. Dev.Immunol. 283649, 2013), IPH-3102 (Basith et al., Exp. Opin. Ther. Pat.21: 927-944, 2011), or a variant thereof. In some embodiments, the TLR3agonist is CQ-07001 (Clinquest). In some embodiments, the TLR3 agonistis Ampligen poly(I):poly(C12U) (Hemispherx Biopharma). In someembodiments, the TLR3 agonist is IPH-31XX (Innate Pharma). In someembodiments, the TLR3 agonist is MCT-465-dsRNA (MultiCell Technologies).

TLR4 Agonists

In some embodiments, the TLR agonist specifically binds to and activatesTLR4 (Peri et al., J. Med. Chem. 57(9):3612-3622, 2014). In someembodiments, the TLR4 agonist is bacterial lipopolysaccharide (LPS) or avariant thereof. In some embodiments, the TLR4 agonist is monophosphoryllipid A (MPL, MPLA, GLA, GLA-SE) (Ribi et al., J. Immunol. 6:567-572,1984; Okemoto et al., J. Immunol. 176:1203-1208, 2006; Matzner et al.,Int. J. Cancer 138:1754-1764, 2016; Cauwelaert et al., PLoS One11(1):e0146372, 2016). In some embodiments, the TLR agonist is AS15 orAS02b (Brichard et al., Vaccine 25(Suppl. 2):B61-B71, 2007; Kruit etal., J. Clin. Oncol. 26(Suppl): Abstract 9065, 2008). In someembodiments, the TLR agonist is an aminoalkyl glucosaminide 4-phosphate(e.g., RC-529, Ribi.529, E6020) or a variant thereof (Baldridge et al.,J. Endotoxin Res. 8:453-458, 2002; Morefield et al., Clin. VaccineImmunol. 14: 1499-1504, 2007). In some embodiments, the TLR agonist ispicibanil (OK-432) (Hazim et al., Med. J. Malaysia 71(6):328-330, 2016;Tian et al., Asian Pac J. Cancer Prev. 16(11):4537-4542, 2015; Rebuffiniet al., Dent Rese. J. 9(Suppl. 2):S192-5196, 2012). In some embodiments,the TLR4 agonist is Spirulina complex polysaccharide (Kwanishi et al.,Microbiol. Immunol. 57:63-73, 2013). In some embodiments, the TLR4agonist is chitohexaose or a variant thereof (Panda et al., 8:e1002717,2012; Barman et al., Cell Death Dis. 7:e2224, 2016). In someembodiments, the TLR4 agonist is E5564 (Eritoran) (Eisai). In someembodiments, the TLR4 agonist is CRX-675 or CRX-527 (GSK).

TLR5 Agonists

In some embodiments, the TLR agonist binds and activates TLR5. In someembodiments, the TLR5 agonist is flagellin or a variant thereof (e.g.,entolimod (CBLB502)) (Yoon et al., Science 335: 859-864, 2012; Fukuzawaet al., J. Immunol. 187:3831-3839, 2011; Brackett et al., PNAS113(7):E874-E883, 2015; Leigh et al., PLoS One 9(1):e85587, 2014;Hossain et al., Blood 120:255, 2012). In some embodiments, the TLR5agonist is flagellin HuHa (Vaxinate) or flagellin HuM2e (Vaxinate).

TLR7/8 Agonists

In some embodiments, the TLR agonist binds and activates TLR7/8 (e.g.,TLR7 agonist, TLR8 agonist, or a TLR7 and TLR8 agonist). In someembodiments, the TLR7/8 agonist is ANA975 (isotorabine)(Anadys/Novartis), ANA773 (Anadys/Novartis),

In some embodiments, the TLR7/8 agonist is an imidazoquinoline or avariant thereof (e.g., imiquimod (Aldara™; Kaspari et al., British J.Dermatology 147: 757-759, 2002; Smorlesi et al., Gene Therapy 12:1324-133, 2005; Prins et al., J. Immunol. 176: 157-164, 2006; Shackletonet al., Cancer Immun. 4:9, 2004; Green et al., Br. J. Dermatol.156(2):337-345, 2007; Geisse et al., Am. Acad. Dermatol. 50(5):722-733,2004; Wolf et al., Arch. Dermatol. 139(3):273-276, 2003), resiquimod(R848; Hemmi et al., Nat. Immunol. 3:196-200, 2002; Jurk et al., Nat.Immunol. 3:49, 2002; Rook et al., Blood 126(12):1452-1461, 2015; Dovediet al., Blood 121: 251-259, 2013). In some embodiments, the TLR agonistis a synthetic imiadzoquinoline mimicking viral single stranded RNA(ssRNA) (852A) or a variant thereof (Dudek et al., Clin. Cancer Res.13(23):7119-7125, 2007; Dummer et al., Clin. Cancer Res. 14(3):856-864,2008; Weigel et al., Am. J. Hematol. 87(10):953-956, 2012; Geller etal., Cancer Immunol. Immunother. 59(12):1877-1884, 2010; Inglefield etal., J. Interferon Cytokine Res. 28(4):253-263, 2008). In someembodiments, the TLR agonist is a small molecule. In some embodiments,the small molecule mimics viral ssRNA (e.g., motolimod (VTX-2337)) or avariant thereof (Dietsch et al., Clin. Cancer Res. 21(24):5445-5452,2015; Northfelt et al., Clin. Cancer Res. 20(14):3683-3691, 2014; Lu etal., Clin. Cancer Res. 18(2):499-509, 2012). In some embodiments, thesmall molecule is GS-9620 or a variant thereof (Bam et al., AntimicrobAgents Chemother. 61(1):e01369, 2016; Rebbapragada et al., PLoS One11(1):e0146835, 2016; Gane et al., J. Hepatol. 63(2): 320-328, 2015;Fosdick et al., J. Med. Chem. 56(18):7324-7333, 2013). In someembodiments, the small molecule is SC1 (Wiedemann et al., Oncoimmunology5(7):e1189051, 2016; Hamm et al., J. Immunol. 6(4):257-265, 2009). Insome embodiments, the small molecule is gardiquimod (Ma et al., Cell.Mol. Immunol. 7:381-388, 2010; Hjelm et al., Hum. Vaccin. Immunother.10(2): 410-416, 2014; Buitendijk et al., AIDS Res. Hum. Retroviruses29(6):907-918, 2013), CL075 (Philbin et al., J. Allergy Clin. Immunol.130:195-204, 2012; Dowling et al., PLoS One 8(3): e58164, 2013), CL097(Gorden et al., J. Immunol. 174:1259-1268, 2005; Gorski et al., Int.Immunol. 18:1115, 2006; Levy et al., Blood 108:1284-1289, 2006;Wille-Reece et al., J. Exp. Med. 203: 1249-1258, 2006), loxoribine (Popeet al., Cell Immunol. 162:333, 1995; Heil et al., Eur. J. Immunol.33:2987-2997, 2003; Lee et al., PNAS 100:6646-6651, 2003), or VTX-294(Dowling et al., PLoS One 8(3):e58164, 2013). In some embodiments, theTLR7/8 agonist is IMO-9200. In some embodiments, the TLR7 agaonist isIPH-32XX (Innate Pharma).

TLR9 Agonists

In some embodiments, the TLR agonist binds and activates TLR9. In someembodiments, the TLR9 agonist is a synthetic oligonucleotide. In someembodiments, the synthetic oligonucleotide contains unmethylated CpGdinucleotide (CpG-ODN) (Krieg, J. Clin. Invest. 117:1184-1194, 2007;Carpentier et al., Neuro-oncol. 8(1):60-66, 2006; Link et al., J.Immunother. 29(5): 558-568, 2006; Pashenkov et al., J. Clin. Oncol.24(36): 5716-5724, 2006; Meng et al., BMC Biotechnol. 11:88, 2011). Insome embodiments, the TLR9 agonist is PF-3512676 or a variant thereof(Hofmann et al., J. Immunother. 31(5):520-527, 2008; Molenkamp et al.,Clin. Caner. Res. 14(14):4532-4542, 2008). In some embodiments, the TLR9agonist is IMO-2055 (EMD1201801) or a variant thereof (Machiels et al.,Investig. New Drugs 31:1207-1216, 2013). In some embodiments, the TLR9agonist is DIMS0150 (Atreya et al., J. Crohns Colitis 10(11):1294-1302,2016). In some embodiments, the TLR9 agonist is CpG7909 (Vaximmune)(Coley, GSK, Novartis, DARPA). In some embodiments, the TLR9 agonist isIMO-9200. In some embodiments, the TLR9 agonist is AVE0675 (Coley,Sanofi Aventis). In some embodiments, the TLR9 agonist is Amplivax(Idera).

Microbial Products as TLR Agonists

In some embodiments, the TLR agonist is a bacterial or viral component.In some embodiments, the TLR agonist is derived from the cell wallMycobacterium bovis (BCG). In some embodiments, the Mycobacterium boviscell wall component is a TLR2 and/or TLR4 agonist (e.g., SMP105 (Murataet al., Cancer Sci. 99:1435-1440, 2008; Miyauchi et al., Drug Discov.Ther. 6: 218-225, 2013; Tsuji et al., Infect Immun. 68: 6883-6890, 2000;Smith et al., Cancer Immunol. Immunother. 63(8):787-796, 2014).Additional examples of TLR agonists are known in the art.

TLR Antagonists

By the term “TLR antagonist” means an agent that decreases the bindingof a TLR agonist to TLR4 or TLR9 expressed in a mammalian cell (e.g., ahuman cell). In some embodiments, any of the compositions, devices, orkits described herein can include a TLR antagonist. For example, a TLRantagonist can be a TLR4 antagonist. In other examples, a TLR antagonistis a TLR9 antagonist. Non-limiting examples of TLR antagonists aredescribed in Fukata et al., Mucosal Immunity 6:451-463, 2013.

A non-limiting example of a TLR4 antagonist is 1A6 (Ungaro et al., Am.J. Physiol. Gastrointest. Liver Physiol. 296:G1167-G1179, 2009) orCRX-526 (Fort et al., J. Immunol. 174:6416-6423, 2005). Additionalexamples of TLR4 antagonists include eritoran tetrasodium (E5564) (Sunet al., Investigative Ophthalmol. Visual Sci. 50(3):1247-1254, 2009),small heat shock protein B8 (HSP22) (Roelofs et al., J. Immunol.176(11):7021-7027, 2006), CRX-527 (Bazin et al., Bioorganic Med. Chem.Letters 18(2):5350-5354, 2008), E5564 (Kitazawa et al., J. Gastroentrol.Hepatol. 25(5):1009-1012, 2010), IAXO-102 (Huggins et al.,Atherosclerosis 242(2):563-570, 2015), AG-411 (Kondo et al., TrendsImmunol. 33(9):449-458, 2012), CRX-52624 (Alderson et al., J. EndotoxinRes. 12(5):313-319, 2006), E5531 (Becker et al., Toxicol. Appl.Pharmacol. 207(2):269-275, 2005).

A non-limiting example of a TLR9 antagonist is adenoviraloligodeoxynucleotides (AV-ODN) (Obermeier et al., Gastroenterology129:913-927, 2005). Additional examples of TLR9 antagonists include ODN2088, ODN 4084-F, ODN INH-1, ODN INH-18, ODN TTAGGG (A151), and G-ODN(each commercially available from InvivoGen). In some embodiments, theTLR9 antagonist is CpG-ODN c41 (Li et al., Vaccine 29:2193-2198, 2011).In some embodiments, the TLR9 antagonist is COV08-0064 (Shaker et al.,Biochemical Pharmacol. 112:90-101, 2016; Hoque et al., J. Immunol.190(8):4297-4304, 2013); ODN 1585, ODN 1826, ODN 2395, and ODN 2088(Boivin et al., Antiviral Res. 96(3):414-421, 2012); IMO-8400 (Zhu etal., J. Immunol. 188(1):119, 2012); IRS869 (Mandl et al., Nature Med.14(10:1077-1087, 2008); IMO-3100 (Hennessy et al., Nature Rev. DrugDiscov. 9(4):293-307, 2010); TTAGGG (Carvalho et al., PLoS One6(11):e28256, 2011); and CpG ODN 2088 (David et al., J. Neurotrauma31(21):1800-1806, 2014).

In some embodiments, the TLR modulator is BL-7040. In some embodiments,the TLR modulator is EN-101. In some embodiments, the TLR modulator isMonarsen.

Endoscopes, Ingestible Devices, and Reservoirs

As discussed herein, in some embodiments, a method of treating a diseaseof the gastrointestinal tract comprises administering to the subject apharmaceutical formulation wherein the pharmaceutical formulation isdelivered proximate to one or more sites of disease by one of variousmethods. For example, the pharmaceutical formulation may be deliveredvia a medical device such as an endoscope, ingestible device, orreservoir; the pharmaceutical formulation may be a solid dosage form, aliquid dosage form, a suppository or an enema for rectal administrationwith different types of release such as sustained or delayed release.

In one embodiment, the pharmaceutical formulation is delivered proximateto one or more sites of disease by an endoscope, ingestible device, orreservoir containing the pharmaceutical formulation.

The GI tract can be imaged using endoscopes, or more recently, byingestible devices that are swallowed. Direct visualization of the GImucosa is useful to detect subtle mucosal alterations, as ininflammatory bowel diseases, as well as any flat or sessile lesions.

As discussed herein, in some embodiments, the method of treating adisease of the gastrointestinal tract comprises administering to thesubject a pharmaceutical formulation. In some embodiments, thepharmaceutical formulation is delivered proximate to one or more sitesof disease by one of various methods. For example, the pharmaceuticalformulation may be delivered via a medical device such as an endoscope,ingestible device, or reservoir; the pharmaceutical formulation may be asolid dosage form, a liquid dosage form, a suppository or an enema forrectal administration with different types of release such as sustainedor delayed release.

In one embodiment, the pharmaceutical formulation is delivered proximateto one or more sites of disease by an endoscope, ingestible device, orreservoir containing the pharmaceutical formulation.

The technology behind standard colonoscopy consists of a long,semi-rigid insertion tube with a steerable tip (stiff if compared to thecolon), which is pushed by the physician from the outside. However,invasiveness, patient discomfort, fear of pain, and—more often thannot—the need for conscious sedation limit the take-up of screeningcolonoscopy. Diagnosis and treatment in the GI tract are dominated bythe use of flexible endoscopes. A few large companies, namely OlympusMedical Systems Co. (Tokyo, Japan), Pentax Medical Co. (Montvale, N.J.,USA), Fujinon, Inc. (Wayne, N.J., USA) and Karl Storz GmbH & Co. KG(Tuttlingen, Germany), cover the majority of the market in flexible GIendoscopy.

Endoscopes may comprise a catheter. As an example, the catheter may be aspray catheter. As an example, a spray catheter may be used to deliverdyes for diagnostic purposes. As an example, a spray catheter may beused to deliver a therapeutic agent at the site of disease in the GItract. For example, the Olypmus PW-205V is a ready-to-use spray catheterthat enables efficient spraying for maximal differentiation of tissuestructures during endoscopy, but may also be used to deliver drugsdiseased tissue.

In a review of robotic endoscopic capsules, Journal of Micro-BioRobotics 11.1-4 (2016): 1-18, Ciuti et al. state that progress inmicro-electromechanical systems (MEMS) technologies have led to thedevelopment of new endoscopic capsules with enhanced diagnosticcapabilities, in addition to traditional visualization of mucosa(embedding, e.g. pressure, pH, blood detection and temperature sensors).

Endoscopic capsules, however, do not have the capability of accuratelylocating a site autonomously. They require doctor oversight over aperiod of hours in order to manually determine the location. Autonomousingestible devices are advantageous in that regard.

Ingestible devices are also advantageous over spray catheters in thatthey are less invasive, thereby allowing for regular dosing morefrequently than spray catheters. Another advantage of ingestible devicesis the greater ease with which they can access, relative to a catheter,certain sections of the GI tract such as the ascending colon, the cecum,and all portions of the small intestine.

Methods and Mechanisms for Localization

In addition to, or as an alternative, to directly visualizing the GItract, one or more different mechanisms can be used to determine thelocation of an ingestible device within the GI tract. Variousimplementations may be used for localization of ingestible deviceswithin the GI tract.

For example, various implementations may be used for localization ofingestible devices within the GI tract. For example, certainimplementations can include one or more electromagnetic sensor coils,magnetic fields, electromagnetic waves, electric potential values,ultrasound positioning systems, gamma scintigraphy techniques or otherradio-tracker technology have been described by others. Alternatively,imaging can be used to localize, for example, using anatomical landmarksor more complex algorithms for 3D reconstruction based on multipleimages. Other technologies rely on radio frequency, which relies onsensors placed externally on the body to receive the strength of signalsemitted by the capsule. Ingestible devices may also be localized basedon reflected light in the medium surrounding the device; pH;temperature; time following ingestion; and/or acoustic signals.

The disclosure provides an ingestible device, as well as related systemsand methods that provide for determining the position of the ingestibledevice within the GI tract of a subject with very high accuracy. In someembodiments, the ingestible device can autonomously determine itsposition within the GI tract of the subject.

Typically, the ingestible device includes one or more processingdevices, and one more machine readable hardware storage devices. In someembodiments, the one or more machine readable hardware storage devicesstore instructions that are executable by the one or more processingdevices to determine the location of the ingestible device in a portionof a GI tract of the subject. In certain embodiments, the one or moremachine readable hardware storage devices store instructions that areexecutable by the one or more processing devices to transmit data to anexternal device (e.g., a base station external to the subject, such as abase station carried on an article worn by the subject) capable ofimplementing the data to determine the location of the device within theGI tract of the subject.

In some embodiments, the location of the ingestible device within the GItract of the subject can be determined to an accuracy of at least 85%,e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least99%, 100%. In some embodiments, the location of the ingestible devicewithin the GI tract of the subject can be determined to an accuracy ofat least 85%, e.g., at least 90%, at least 95%, at least 97%, at least98%, at least 99%, 100%. In such embodiments, the portion of the GItract of the subject can include, for example, the esophagus, thestomach, duodenum, the jejunum, and/or the terminal ileum, cecum andcolon. An exemplary and non-limiting embodiment is provided below inExample 13.

In certain embodiments, the location of the ingestible device within theesophagus of the subject can be determined to an accuracy of at least85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, 100%. An exemplary and non-limiting embodiment is providedbelow in Example 13.

In some embodiments, the location of the ingestible device within thestomach of the subject can be determined to an accuracy of at least 85%,e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least99%, 100%. An exemplary and non-limiting embodiment is provided below inExample 13.

In certain embodiments, the location of the ingestible device within theduodenum of the subject can be determined to an accuracy of at least85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, 100%. An exemplary and non-limiting embodiment is providedbelow in Example 13.

In some embodiments, the location of the ingestible device within thejejunum of the subject can be determined to an accuracy of at least 85%,e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least99%, 100%. An exemplary and non-limiting embodiment is provided below inExample 13.

In certain embodiments, the location of the ingestible device within theterminal ileum, cecum and colon of the subject can be determined to anaccuracy of at least 85%, e.g., at least 90%, at least 95%, at least97%, at least 98%, at least 99%, 100%.

In some embodiments, the location of the ingestible device within thececum of the subject can be determined to an accuracy of at least 85%,e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least99%, 100%. An exemplary and non-limiting embodiment is provided below inExample 13. In such embodiments, the portion of the portion of the GItract of the subject can include, for example, the esophagus, thestomach, duodenum, the jejunum, and/or the terminal ileum, cecum andcolon.

In certain embodiments, the location of the ingestible device within theesophagus of the subject can be determined to an accuracy of at least85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, 100%.

In some embodiments, the location of the ingestible device within thestomach of the subject can be determined to an accuracy of at least 85%,e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least99%, 100%.

In certain embodiments, the location of the ingestible device within theduodenum of the subject can be determined to an accuracy of at least85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, 100%.

In some embodiments, the location of the ingestible device within thejejunum of the subject can be determined to an accuracy of at least 85%,e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least99%, 100%.

In certain embodiments, the location of the ingestible device within theterminal ileum, cecum and colon of the subject can be determined to anaccuracy of at least 85%, e.g., at least 90%, at least 95%, at least97%, at least 98%, at least 99%, 100%.

In some embodiments, the location of the ingestible device within thececum of the subject can be determined to an accuracy of at least 85%,e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least99%, 100%.

As used herein, the term “reflectance” refers to a value derived fromlight emitted by the device, reflected back to the device, and receivedby a detector in or on the device. For example, in some embodiments thisrefers to light emitted by the device, wherein a portion of the light isreflected by a surface external to the device, and the light is receivedby a detector located in or on the device.

As used herein, the term “illumination” refers to any electromagneticemission. In some embodiments, an illumination may be within the rangeof Infrared Light (IR), the visible spectrum and ultraviolet light (UV),and an illumination may have a majority of its power centered at aparticular wavelength in the range of 100 nm to 1000 nm. In someembodiments, it may be advantageous to use an illumination with amajority of its power limited to one of the infrared (750 nm-1000 nm),red (600 nm-750 nm), green (495 nm-600 nm), blue (400 nm-495 nm), orultraviolet (100 nm-400 nm) spectrums. In some embodiments a pluralityof illuminations with different wavelengths may be used. Forillustrative purposes, the embodiments described herein may refer to theuse of green or blue spectrums of light. However, it is understood thatthese embodiments may use any suitable light having a wavelength that issubstantially or approximately within the green or blue spectra definedabove, and the localization systems and methods described herein may useany suitable spectra of light.

Referring now to FIG. 1, shown therein is a view of an exampleembodiment of an ingestible device 100, which may be used to identify alocation within a gastrointestinal (GI) tract. In some embodiments,ingestible device 100 may be configured to autonomously determinewhether it is located in the stomach, a particular portion of the smallintestine such as a duodenum, jejunum, or ileum, or the large intestineby utilizing sensors operating with different wavelengths of light.Additionally, ingestible device 100 may be configured to autonomouslydetermine whether it is located within certain portions of the smallintestine or large intestine, such as the duodenum, the jejunum, thececum, or the colon.

Ingestible device 100 may have a housing 102 shaped similar to a pill orcapsule. The housing 102 of ingestible device 100 may have a first endportion 104, and a second end portion 106. The first end portion 104 mayinclude a first wall portion 108, and second end portion 106 may includea second wall portion 110. In some embodiments, first end portion 104and second end portion 106 of ingestible device 100 may be manufacturedseparately, and may be affixed together by a connecting portion 112.

In some embodiments, ingestible device 100 may include an opticallytransparent window 114. Optically transparent window 114 may betransparent to various types of illumination in the visible spectrum,infrared spectrum, or ultraviolet light spectrum, and ingestible device100 may have various sensors and illuminators located within the housing102, and behind the transparent window 114. This may allow ingestibledevice 100 to be configured to transmit illumination at differentwavelengths through transparent window 114 to an environment external tohousing 102 of ingestible device 100, and to detect a reflectance from aportion of the illumination that is reflected back through transparentwindow 114 from the environment external to housing 102. Ingestibledevice 100 may then use the detected level of reflectance in order todetermine a location of ingestible device 100 within a GI tract. In someembodiments, optically transparent window 114 may be of any shape andsize, and may wrap around the circumference of ingestible device 100. Inthis case, ingestible device 100 may have multiple sets of sensors andilluminators positioned at different locations azimuthally behind window114.

In some embodiments, ingestible device 100 may optionally include anopening 116 in the second wall portion 110. In some embodiments, thesecond wall portion 110 may be configured to rotate around thelongitudinal axis of ingestible device 100 (e.g., by means of a suitablemotor or other actuator housed within ingestible device 100). This mayallow ingestible device 100 to obtain a fluid sample from the GI tract,or release a substance into the GI tract, through opening 116.

FIG. 2 shows an exploded view of ingestible device 100. In someembodiments, ingestible device 100 may optionally include a rotationassembly 118. Optional rotation assembly 118 may include a motor 118-1driven by a microcontroller (e.g., a microcontroller coupled to printedcircuit board 120), a rotation position sensing ring 118-2, and astorage sub-unit 118-3 configured to fit snugly within the second endportion 104. In some embodiments, rotation assembly 118 may cause secondend portion 104, and opening 116, to rotate relative to the storagesub-unit 118-3. In some embodiments, there may be cavities on the sideof storage sub-unit 118-3 that function as storage chambers. When theopening 116 is aligned with a cavity on the side of the storage sub-unit118-3, the cavity on the side of the storage sub-unit 118-3 may beexposed to the environment external to the housing 102 of ingestibledevice 100. In some embodiments, the storage sub-unit 118-3 may beloaded with a medicament or other substance prior to the ingestibledevice 100 being administered to a subject. In this case, the medicamentor other substance may be released from the ingestible device 100 byaligning opening 116 with the cavity within storage sub-unit 118-3. Insome embodiments, the storage sub-unit 118-3 may be configured to hold afluid sample obtained from the GI tract. For example, ingestible device100 may be configured to align opening 116 with the cavity withinstorage sub-unit 118-3, thus allowing a fluid sample from the GI tractto enter the cavity within storage sub-unit 118-3. Afterwards,ingestible device 100 may be configured to seal the fluid sample withinstorage sub-unit 118-3 by further rotating the second end portion 106relative to storage sub-unit 118-3. In some embodiments, storagesub-unit 118-3 may also contain a hydrophilic sponge, which may enableingestible device 100 to better draw certain types of fluid samples intoingestible device 100. In some embodiments, ingestible device 100 may beconfigured to either obtain a sample from within the GI tract, or torelease a substance into the GI tract, in response to determining thatingestible device 100 has reached a predetermined location within the GItract. For example, ingestible device 100 may be configured to obtain afluid sample from the GI tract in response to determining that theingestible device has entered the jejunum portion of the small intestine(e.g., as determined by process 900 discussed in relation to FIG. 9).Other ingestible devices capable of obtaining samples or releasingsubstances are discussed in commonly-assigned PCT Application No.PCT/CA2013/000133 filed Feb. 15, 2013, commonly-assigned U.S.Provisional Application No. 62/385,553, and commonly-assigned U.S.Provisional Application No. 62/376,688, which each are herebyincorporated by reference herein in their entirety. It is understoodthat any suitable method of obtaining samples or releasing substancesmay be incorporated into some of the embodiments of the ingestibledevices disclosed herein, and that the systems and methods fordetermining a location of an ingestible device may be incorporated intoany suitable type of ingestible device.

Ingestible device 100 may include a printed circuit board (PCB) 120, anda battery 128 configured to power PCB 120. PCB 120 may include aprogrammable microcontroller, and control and memory circuitry forholding and executing firmware or software for coordinating theoperation of ingestible device 100, and the various components ofingestible device 100. For example, PCB 120 may include memory circuitryfor storing data, such as data sets of measurements collected by sensingsub-unit 126, or instructions to be executed by control circuitry toimplement a localization process, such as, for example, one or more ofthe processes, discussed herein, including those discussed below inconnection with one or more of the associated flow charts. PCB 120 mayinclude a detector 122 and an illuminator 124, which together formsensing sub-unit 126. In some embodiments, control circuitry within PCB120 may include processing units, communication circuitry, or any othersuitable type of circuitry for operating ingestible device 100. Forillustrative purposes, only a single detector 122 and a singleilluminator 124 forming a single sensing sub-unit 126 are shown.However, it is understood that in some embodiments there may be multiplesensing sub-units, each with a separate illuminator and detector, withiningestible device 100. For example, there may be several sensingsub-units spaced azimuthally around the circumference of the PCB 120,which may enable ingestible device 100 to transmit illumination anddetect reflectances or ambient light in all directions around thecircumference of the device. In some embodiments, sensing sub-unit 126may be configured to generate an illumination using illuminator 124,which is directed through the window 114 in a radial direction away fromingestible device 100. This illumination may reflect off of theenvironment external to ingestible device 100, and the reflected lightcoming back into ingestible device 100 through window 114 may bedetected as a reflectance by detector 122.

In some embodiments, window 114 may be of any suitable shape and size.For example, window 114 may extend around a full circumference ofingestible device 100. In some embodiments there may be a plurality ofsensing sub-units (e.g., similar to sensing sub-unit 126) located atdifferent positions behind the window. For example, three sensingsub-units may be positioned behind the window at the same longitudinallocation, but spaced 120 degrees apart azimuthally. This may enableingestible device 100 to transmit illuminations in all directionsradially around ingestible device 100, and to measure each of thecorresponding reflectances.

In some embodiments, illuminator 124 may be capable of producingillumination at a variety of different wavelengths in the ultraviolet,infrared, or visible spectrum. For example, illuminator 124 may beimplemented by using Red-Green-Blue Light-Emitting diode packages(RGB-LED). These types of RGB-LED packages are able to transmit red,blue, or green illumination, or combinations of red, blue, or greenillumination. Similarly, detector 122 may be configured to sensereflected light of the same wavelengths as the illumination produced byilluminator 124. For example, if illuminator 124 is configured toproduce red, blue, or green illumination, detector 122 may be configuredto detect different reflectances produced by red, blue, or greenillumination (e.g., through the use of an appropriately configuredphotodiode). These detected reflectances may be stored by ingestibledevice 100 (e.g., within memory circuitry of PCB 120), and may then beused by ingestible device 100 in determining a location of ingestibledevice 100 within the GI tract (e.g., through the use of process 500(FIG. 5), process 600 (FIG. 6), or process 900 (FIG. 9)).

It is understood that ingestible device 100 is intended to beillustrative, and not limiting. It will be understood that modificationsto the general shape and structure of the various devices and mechanismsdescribed in relation to FIG. 1 and FIG. 2 may be made withoutsignificantly changing the functions and operations of the devices andmechanisms. For example, ingestible device 100 may have a housing formedfrom a single piece of molded plastic, rather than being divided into afirst end portion 104 and a second end portion 106. As an alternateexample, the location of window 114 within ingestible device 100 may bemoved to some other location, such as the center of ingestible device100, or to one of the ends of ingestible device 100. Moreover, thesystems and methods discussed in relation to FIGS. 1-10 may beimplemented on any suitable type of ingestible device, provided that theingestible device is capable of detecting reflectances or levels ofillumination in some capacity. For example, in some embodimentsingestible device 100 may be modified to replace detector 122 with animage sensor, and the ingestible device may be configured to measurerelative levels of red, blue, or green light by decomposing a recordedimage into its individual spectral components. Other examples ofingestible devices with localization capabilities, which may be utilizedin order to implement the systems and methods discussed in relation toFIG. 1-11, are discussed in co-owned PCT Application No.PCT/US2015/052500 filed on Sep. 25, 2015, which is hereby incorporatedby reference herein in its entirety. Furthermore, it should be notedthat the features and limitations described in any one embodiment may beapplied to any other embodiment herein, and the descriptions andexamples relating to one embodiment may be combined with any otherembodiment in a suitable manner.

FIG. 3 is a diagram of an ingestible device during an example transitthrough a gastrointestinal (GI) tract, in accordance with someembodiments of the disclosure. Ingestible device 300 may include anyportion of any other ingestible device discussed in this disclosure(e.g., ingestible device 100 (FIG. 1)), and may be any suitable type ofingestible device with localization capabilities. For example,ingestible device 300 may be one embodiment of ingestible device 100without the optional opening 116 (FIG. 1) or optional rotation assembly118 (FIG. 2)). In some embodiments, ingestible device 300 may beingested by a subject, and as ingestible device 300 traverses the GItract, ingestible device 300 may be configured to determine its locationwithin the GI tract. For example, the movement of ingestible device 300and the amount of light detected by ingestible device 300 (e.g., viadetector 122 (FIG. 2)) may vary substantially depending on the locationof ingestible device 300 within the GI tract, and ingestible device 300may be configured to use this information to determine a location ofingestible device 300 within the GI tract. For instance, ingestibledevice 300 may detect ambient light from the surrounding environment, orreflectances based on illumination generated by ingestible device 300(e.g., generated by illuminator 124 (FIG. 1)), and use this informationto determine a location of ingestible device 300 through processes, suchas described herein. The current location of ingestible device 300, andthe time that ingestible device 300 detected each transition between thevarious portions of the GI tract, may then be stored by ingestibledevice 300 (e.g., in memory circuitry of PCB 120 (FIG. 2)), and may beused for any suitable purpose.

Shortly after ingestible device 300 is ingested, ingestible device willtraverse the esophagus 302, which may connect the subject's mouth to astomach 306. In some embodiments, ingestible device 300 may beconfigured to determine that it has entered the esophagus portion GItract by measuring the amount and type of light (e.g., via detector 122(FIG. 2)) in the environment surrounding the ingestible device 300. Forinstance, ingestible device 300 may detect higher levels of light in thevisible spectrum (e.g., via detector 122 (FIG. 2)) while outside thesubject's body, as compared to the levels of light detected while withinthe GI tract. In some embodiments, ingestible device 300 may havepreviously stored data (e.g., on memory circuitry of PCB 120 (FIG. 2))indicating a typical level of light detected when outside of the body,and the ingestible device 300 may be configured to determine that entryto the body has occurred when a detected level of light (e.g., detectedvia detector 122 (FIG. 2)) has been reduced beyond a threshold level(e.g., at least a 20-30% reduction) for a sufficient period of time(e.g., 5.0 seconds).

In some embodiments, ingestible device 300 may be configured to detect atransition from esophagus 302 to stomach 306 by passing throughsphincter 304. In some embodiments, ingestible device 300 may beconfigured to determine whether it has entered stomach 306 based atleast in part on a plurality of parameters, such as but not limited tothe use of light or temperature measurements (e.g., via detector 122(FIG. 2) or via a thermometer within ingestible device 300), pHmeasurements (e.g., via a pH meter within ingestible device 300), timemeasurements (e.g., as detected through the use of clock circuitryincluded within PCB 120 (FIG. 2)), or any other suitable information.For instance, ingestible device 300 may be configured to determine thatingestible device 300 has entered stomach 306 after detecting that ameasured temperature of ingestible device 300 exceeds 31 degreesCelsius. Additionally, or alternately, ingestible device 300 may beconfigured to automatically determine it has entered stomach 306 afterone minute (or another pre-set time duration parameter, 80 seconds, 90seconds, etc.) has elapsed from the time that ingestible device 300 wasingested, or one minute (or another pre-set time duration parameter, 80seconds, 90 seconds, etc.) from the time that ingestible device 300detected that it has entered the GI tract.

Stomach 306 is a relatively large, open, and cavernous organ, andtherefore ingestible device 300 may have a relatively large range ofmotion. By comparison, the motion of ingestible device 300 is relativelyrestricted within the tube-like structure of the duodenum 310, thejejunum 314, and the ileum (not shown), all of which collectively formthe small intestine. Additionally, the interior of stomach 306 hasdistinct optical properties from duodenum 310 and jejunum 314, which mayenable ingestible device 300 to detect a transition from stomach 306 toduodenum 310 through the appropriate use of measured reflectances (e.g.,through the use of reflectances measured by detector 122 (FIG. 2)), asused in conjunction with process 600 (FIG. 6)).

In some embodiments, ingestible device 300 may be configured to detect apyloric transition from stomach 306 to duodenum 310 through the pylorus308. For instance, in some embodiments, ingestible device 300 may beconfigured to periodically generate illumination in the green and bluewavelengths (e.g., via illuminator 124 (FIG. 2)), and measure theresulting reflectances (e.g., via detector 122 (FIG. 2)). Ingestibledevice 300 may be configured to then use a ratio of the detected greenreflectance to the detected blue reflectance to determine whetheringestible device 300 is located within the stomach 306, or duodenum 310(e.g., via process 600 (FIG. 6)). In turn, this may enable ingestibledevice 300 to detect a pyloric transition from stomach 306 to duodenum310, an example of which is discussed in relation to FIG. 6.

Similarly, in some embodiments, ingestible device 300 may be configuredto detect a reverse pyloric transition from duodenum 310 to stomach 306.Ingestible device 300 will typically transition naturally from stomach306 to duodenum 310, and onward to jejunum 314 and the remainder of theGI tract. However, similar to other ingested substances, ingestibledevice 300 may occasionally transition from duodenum 310 back to stomach306 as a result of motion of the subject, or due to the natural behaviorof the organs with the GI tract. To accommodate this possibility,ingestible device 300 may be configured to continue to periodicallygenerate illumination in the green and blue wavelengths (e.g., viailluminator 124 (FIG. 2)), and measure the resulting reflectances (e.g.,via detector 122 (FIG. 2)) to detect whether or not ingestible device300 has returned to stomach 306. An exemplary detection process isdescribed in additional detail in relation to FIG. 6.

After entering duodenum 310, ingestible device 300 may be configured todetect a transition to the jejunum 314 through the duodenojejunalflexure 312. For example, ingestible device 300 may be configured to usereflectances to detect peristaltic waves within the jejunum 314, causedby the contraction of the smooth muscle tissue lining the walls of thejejunum 314. In particular, ingestible device 300 may be configured tobegin periodically transmitting illumination (and measuring theresulting reflectances (e.g., via detector 122 and illuminator 124 ofsensing sub-unit 126 (FIG. 2)) at a sufficiently high frequency in orderto detect muscle contractions within the jejunum 314. Ingestible device300 may then determine that it has entered the jejunum 314 in responseto having detected either a first muscle contraction, or a predeterminednumber of muscle contractions (e.g., after having detected three musclecontractions in sequence). The interaction of ingestible device 300 withthe walls of jejunum 314 is also discussed in relation to FIG. 4, and anexample of this detection process is described in additional detail inrelation to FIG. 9.

FIG. 4 is a diagram of an ingestible device during an example transitthrough a jejunum, in accordance with some embodiments of thedisclosure. Diagrams 410, 420, 430, and 440 depict ingestible device 400as it traverses through a jejunum (e.g., jejunum 314), and howingestible device 400 interacts with peristaltic waves formed by walls406A and 406B (collectively, walls 406) of the jejunum. In someimplementations, ingestible device 400 may include any portion of anyother ingestible device discussed in this disclosure (e.g., ingestibledevice 100 (FIG. 1) or ingestible device 300 (FIG. 3)), and may be anysuitable type of ingestible device with localization capabilities. Forexample, ingestible device 400 may be substantially similar to theingestible device 300 (FIG. 3) or ingestible device 100 (FIG. 1), withwindow 404 being the same as window 114 (FIG. 1), and sensing sub-unit402 being the same as sensing sub-unit 126 (FIG. 2).

Diagram 410 depicts ingestible device 400 within the jejunum, when thewalls 406 of the jejunum are relaxed. In some embodiments, the confinedtube-like structure of the jejunum naturally causes ingestible device400 to be oriented longitudinally along the length of the jejunum, withwindow 404 facing walls 406. In this orientation, ingestible device 400may use sensing sub-unit 402 to generate illumination (e.g., viailluminator 124 (FIG. 2)) oriented towards walls 406, and to detect theresulting reflectances (e.g., via detector 122 (FIG. 2)) from theportion of the illumination reflected off of walls 406 and back throughwindow 404. In some embodiments, ingestible device 400 may be configuredto use sensing sub-unit 402 to generate illumination and measure theresulting reflectance with sufficient frequency to detect peristalticwaves within the jejunum. For instance, in a healthy human subject,peristaltic waves may occur at a rate of approximately 0.1 Hz to 0.2 Hz.Therefore, the ingestible device 400 may be configured to generateillumination and measure the resulting reflectance at least once every2.5 seconds (i.e., the minimum rate necessary to detect a 0.2 Hzsignal), and preferably at a higher rate, such as once every 0.5seconds, which may improve the overall reliability of the detectionprocess due to more data points being available. It is understood thatthe ingestible device 400 need not gather measurements at preciseintervals, and in some embodiments the ingestible device 400 may beadapted to analyze data gathered at more irregular intervals, providedthat there are still a sufficient number of appropriately spaced datapoints to detect 0.1 Hz to 0.2 Hz signals.

Diagram 420 depicts ingestible device 400 within the jejunum, when thewalls 406 of the jejunum begin to contract and form a peristaltic wave.Diagram 420 depicts contracting portion 408A of wall 406A andcontracting portion 408B of wall 406B (collectively, contracting portion408 of wall 406) that form a peristaltic wave within the jejunum. Theperistaltic wave proceeds along the length of the jejunum as differentportions of wall 406 contract and relax, causing it to appear as ifcontracting portions 408 of wall 406 proceed along the length of thejejunum (i.e., as depicted by contracting portions 408 proceeding fromleft to right in diagrams 410-430). While in this position, ingestibledevice 400 may detect a similar level of reflectance (e.g., through theuse of illuminator 124 and detector 122 of sensing sub-unit 126 (FIG.2)) as detected when there is no peristaltic wave occurring (e.g., asdetected when ingestible device 400 is in the position indicated indiagram 410).

Diagram 430 depicts ingestible device 400 within the jejunum, when thewalls 406 of the jejunum continue to contract, squeezing aroundingestible device 400. As the peristaltic wave proceeds along the lengthof the jejunum, contracting portions 408 of wall 406 may squeeze tightlyaround ingestible device 400, bringing the inner surface of wall 406into contact with window 404. While in this position, ingestible device400 may detect a change in a reflectance detected as a result ofillumination produced by sensing sub-unit 402. The absolute value of thechange in the measured reflectance may depend on several factors, suchas the optical properties of the window 404, the spectral components ofthe illumination, and the optical properties of the walls 406. However,ingestible device 400 may be configured to store a data set with thereflectance values over time, and search for periodic changes in thedata set consistent with the frequency of the peristaltic waves (e.g.,by analyzing the data set in the frequency domain, and searching forpeaks between 0.1 Hz to 0.2 Hz). This may enable ingestible device 400to detect muscle contractions due to peristaltic waves withoutforeknowledge of the exact changes in reflectance signal amplitude thatmay occur as a result of detecting the muscle contractions of theperistaltic wave. An example procedure for detecting muscle contractionsis discussed further in relation to FIG. 9, and an example of areflectance data set gathered while ingestible device 400 is locatedwithin the jejunum is discussed in relation to FIG. 10.

Diagram 440 depicts ingestible device 400 within the jejunum, when theperistaltic wave has moved past ingestible device 400. Diagram 440depicts contracting portions 408 that form the peristaltic wave withinthe jejunum having moved past the end of ingestible device 400. Theperistaltic wave proceeds along the length of the jejunum as differentportions of wall 406 contract and relax, causing it to appear as ifcontracting portions 408 of wall 406 proceed along the length of thejejunum (i.e., as depicted by contracting portions 408 proceeding fromleft to right in diagrams 410-430). While in this position, ingestibledevice 400 may detect a similar level of reflectance (e.g., through theuse of illuminator 124 and detector 122 of sensing sub-unit 126 (FIG.2)) as detected when there is no peristaltic wave occurring (e.g., asdetected when ingestible device 400 is in the position indicated indiagram 410, or diagram 420).

Depending on the species of the subject, peristaltic waves may occurwith relatively predictable regularity. After the peristaltic wave haspassed over ingestible device 400 (e.g., as depicted in diagram 440),the walls 406 of the jejunum may relax again (e.g., as depicted indiagram 410), until the next peristaltic wave begins to form. In someembodiments, ingestible device 400 may be configured to continue togather reflectance value data while it is within the GI tract, and maystore a data set with the reflectance values over time. This may allowingestible device 400 to detect each of the muscle contractions as theperistaltic wave passes over ingestible device 400 (e.g., as depicted indiagram 430), and may enable ingestible device 400 to both count thenumber of muscle contractions that occur, and to determine that acurrent location of the ingestible device 400 is within the jejunum. Forexample, ingestible device 400 may be configured to monitor for possiblemuscle contractions while is inside either the stomach or the duodenum,and may determine that ingestible device 400 has moved to the jejunum inresponse to detecting a muscle contraction consistent with a peristalticwave.

FIG. 5 is a flowchart illustrating some aspects of a localizationprocess used by the ingestible device. Although FIG. 5 may be describedin connection with the ingestible device 100 for illustrative purposes,this is not intended to be limiting, and either portions or the entiretyof the localization procedure 500 described in FIG. 5 may be applied toany device discussed in this application (e.g., the ingestible devices100, 300, and 400), and any of the ingestible devices may be used toperform one or more parts of the process described in FIG. 5.Furthermore, the features of FIG. 5 may be combined with any othersystems, methods or processes described in this application. Forexample, portions of the process in FIG. 5 may be integrated into orcombined with the pyloric transition detection procedure described byFIG. 6, or the jejunum detection process described by FIG. 9.

At 502, the ingestible device (e.g., ingestible device 100, 300, or 400)gathers measurements (e.g., through detector 122 (FIG. 2)) of ambientlight. For example, ingestible device 100 may be configured toperiodically measure (e.g., through detector 122 (FIG. 2)) the level ofambient light in the environment surrounding ingestible device 100. Insome embodiments, the type of ambient light being measured may depend onthe configuration of detector 122 within ingestible device 100. Forexample, if detector 122 is configured to measure red, green, and bluewavelengths of light, ingestible device 100 may be configured to measurethe ambient amount of red, green, and blue light from the surroundingenvironment. In some embodiments, the amount of ambient light measuredby ingestible device 100 will be larger in the area external to the body(e.g., a well-lit room where ingestible device 100 is being administeredto a subject) and in the oral cavity of the subject, as compared to theambient level of light measured by ingestible device 100 when inside ofan esophagus, stomach, or other portion of the GI tract (e.g., esophagus302, stomach 306, duodenum 310, or jejunum 314 (FIG. 3)).

At 504, the ingestible device (e.g., ingestible device 100, 300, or 400)determines (e.g., via control circuitry within PCB 120 (FIG. 2)) whetherthe ingestible device has detected entry into the GI tract. For example,ingestible device 100 may be configured to determine when the mostrecent measurement of ambient light (e.g., the measurement gathered at502) indicates that the ingestible device has entered the GI tract. Forinstance, the first time that ingestible device 100 gatherers ameasurement of ambient light at 502, ingestible device 100 may storethat measurement (e.g., via storage circuitry within PCB 120 (FIG. 2))as a typical level of ambient light external to the body. Ingestibledevice 100 may be configured to then compare the most recent measurementof ambient light to the typical level of ambient light external to thebody (e.g., via control circuitry within PCB 120 (FIG. 2)), anddetermine that ingestible device 100 has entered the GI tract when themost recent measurement of ambient light is substantially smaller thanthe typical level of ambient light external to the body. For example,ingestible device 100 may be configured to detect that it has enteredthe GI tract in response to determining that the most recent measurementof ambient light is less than or equal to 20% of the typical level ofambient light external to the body. If ingestible device 100 determinesthat it has detected entry into the GI tract (e.g., that ingestibledevice 100 has entered at least the esophagus 302 (FIG. 3)), process 500proceeds to 506. Alternately, if ingestible device 100 determines thatit has not detected entry into the GI tract (e.g., as a result of themost recent measurement being similar to the typical level of ambientlight external to the body), process 500 proceeds back to 502 where theingestible device 100 gathers further measurements. For instance,ingestible device 100 may be configured to wait a predetermined amountof time (e.g., five seconds, ten seconds, etc.), and then gather anothermeasurement of the level of ambient light from the environmentsurrounding ingestible device 100.

At 506, the ingestible device (e.g., ingestible device 100, 300, or 400)waits for a transition from the esophagus to the stomach (e.g., fromesophagus 302 to stomach 306 (FIG. 3)). For example, ingestible device100 may be configured to determine that it has entered the stomach(e.g., stomach 306 (FIG. 3)) after waiting a predetermined period oftime after having entered the GI tract. For instance, a typicalesophageal transit time in a human patient may be on the order of 15-30seconds. In this case, after having detected that ingestible device 100has entered the GI tract at 504 (i.e., after detecting that ingestibledevice 100 has reached at least esophagus 302 (FIG. 3)), ingestibledevice 100 may be configured to wait one minute, or a similar amount oftime longer than the typical esophageal transmit time (e.g.,ninety-seconds), before automatically determining that ingestible device100 has entered at least the stomach (e.g., stomach 306 (FIG. 3)).

In some embodiments, the ingestible device (e.g., ingestible device 100,300, or 400) may also determine it has entered the stomach based onmeasurements of pH or temperature. For example, ingestible device 100may be configured to determine that it has entered the stomach if atemperature of ingestible device has increased to at least 31 degreesCelsius (i.e., consistent with the temperature inside the stomach), orif a measured pH of the environment surrounding ingestible device 100 issufficiently acidic (i.e., consistent with the acidic nature of gastricjuices that may be found inside the stomach).

At 508, the ingestible device (e.g., ingestible device 100, 300, or 400)stores data indicating the ingestible device has entered the stomach(e.g., stomach 306 (FIG. 3)). For example, after having waited asufficient amount of time at 506, ingestible device 100 may store data(e.g., within storage circuitry of PCB 120 (FIG. 2)) indicative ofingestible device 100 having entered at least the stomach. Onceingestible device 100 reaches at least the stomach, process 500 proceedsto 510 where ingestible device 100 may be configured to gather data todetect entry into the duodenum (e.g., duodenum 310 (FIG. 3)).

In some embodiments, process 500 may also simultaneously proceed from508 to 520, where ingestible device 100 may be configured to gather datain order to detect muscle contractions and detect entry into the jejunum(e.g., jejunum 314 (FIG. 3)). In some embodiments, ingestible device 100may be configured to simultaneously monitor for entry into the duodenumat 516-518, as well as detect for entry into the jejunum at 520-524.This may allow ingestible device 100 to determine when it has enteredthe jejunum (e.g., as a result of detecting muscle contractions), evenwhen it fails to first detect entry into the duodenum (e.g., as a resultof very quick transit times of the ingestible device through theduodenum).

At 510, the ingestible device (e.g., ingestible device 100, 300, or 400)gathers measurements of green and blue reflectance levels (e.g., throughthe use of illuminator 124 and detector 122 of sensing sub-unit 126(FIG. 2)) while in the stomach (e.g., stomach 306 (FIG. 3)). Forexample, ingestible device 100 may be configured to periodically gathermeasurements of green and blue reflectance levels while in the stomach.For instance, ingestible device 100 may be configured to transmit agreen illumination and a blue illumination (e.g., via illuminator 124(FIG. 2)) every five to fifteen seconds, and measure the resultingreflectance (e.g., via detector 122 (FIG. 2)). Every time thatingestible device 100 gathers a new set of measurements, themeasurements may be added to a stored data set (e.g., stored withinmemory circuitry of PCB 120 (FIG. 2)). The ingestible device 100 maythen use this data set to determine whether or not ingestible device 100is still within a stomach (e.g., stomach 306 (FIG. 3)), or a duodenum(e.g., duodenum 310 (FIG. 3)).

In some embodiments, the ingestible device (e.g., ingestible device 100,300, or 400) may be configured to detect a first reflectance based ongenerating an illumination of a first wavelength in approximately thegreen spectrum of light (between 495-600 nm), and detecting a secondreflectance based on generating an illumination of the second wavelengthin approximately the blue spectrum of light (between 400-495 nm). Insome embodiments, the ingestible device may ensure that the illuminationin the green spectrum and the illumination in the blue spectrum havewavelengths separated by at least 50 nm. This may enable ingestibledevice 100 to sufficiently distinguish between the two wavelengths whendetecting the reflectances (e.g., via detector 122 (FIG. 2)). It isunderstood that the separation of 50 nm is intended to be illustrative,and not limiting, and depending on the accuracy of the detectors withiningestible device 100, smaller separations may be possible to be used.

At 512, the ingestible device (e.g., ingestible device 100, 300, or 400)determines (e.g., using control circuitry within PCB 120 (FIG. 2))whether the ingestible device has detected a transition from the stomach(e.g., stomach 306 (FIG. 3)) to a duodenum (e.g., duodenum 310 (FIG. 3))based on a ratio of green and blue (G/B) reflectance levels. Forexample, ingestible device 100 may obtain (e.g., from memory circuitryof PCB 120 (FIG. 2)) a data set containing historical data for therespective ratio of the green reflectance to the blue reflectance asmeasured at a respective time. Generally speaking, a duodenum (e.g.,duodenum 310 (FIG. 3)) of a human subject reflects a higher ratio ofgreen light to blue light, as compared to the ratio of green light toblue light that is reflected by a stomach (e.g., stomach 306 (FIG. 3)).Based on this, ingestible device 100 may be configured to take a firstset of ratios from the data set, representing the result of recentmeasurements, and compare them to a second set of ratios from the dataset, representing the results of past measurements. When the ingestibledevice 100 determines that the mean value of the first set of ratios issubstantially larger than the mean value of the second set of ratios(i.e., that the ratio of reflected green light to reflected blue lighthas increased), the ingestible device 100 may determine that it hasentered the duodenum (e.g., duodenum 310 (FIG. 3)) from the stomach(e.g., stomach 306 (FIG. 3)). If the ingestible device 100 detects atransition from the stomach (e.g., stomach 306 (FIG. 3)) to a duodenum(e.g., duodenum 310 (FIG. 3)), process 500 proceeds to 514, whereingestible device 100 stores data indicating that the ingestible device100 has entered the duodenum (e.g., duodenum 310 (FIG. 3)).Alternatively, if the ingestible device determines that the ingestibledevice has not transitioned from the stomach (e.g., stomach 306 (FIG.3)) to the duodenum (e.g., duodenum 310 (FIG. 3)), process 500 proceedsback to 510 to gather more measurements of green and blue reflectancelevels while still in the stomach (e.g., stomach 306 (FIG. 3)). Anexample procedure for using measurements of green and blue reflectancesto monitor for transitions between the stomach and the duodenum isdiscussed in greater detail in relation to FIG. 6.

In some embodiments, the first time that ingestible device 100 detects atransition from the stomach (e.g., stomach 306 (FIG. 3)) to the duodenum(e.g., duodenum 310 (FIG. 3)), ingestible device 100 may be configuredto take a mean of the second set of data, (e.g., the set of datapreviously recorded while in stomach 306 (FIG. 3)) and store this as atypical ratio of green light to blue light detected within the stomach(e.g., stomach 306 (FIG. 3)) (e.g., within memory circuitry of PCB 120(FIG. 2)). This stored information may later be used by ingestibledevice 100 to determine when ingestible device 100 re-enters the stomach(e.g., stomach 306 (FIG. 3)) from the duodenum (e.g., duodenum 310 (FIG.3)) as a result of a reverse pyloric transition.

At 514, the ingestible device (e.g., ingestible device 100, 300, or 400)stores data indicating that the ingestible device has entered theduodenum (e.g., duodenum 310 (FIG. 3)). For example, ingestible device100 may store a flag within local memory (e.g., memory circuitry of PCB120) indicating that the ingestible device 100 is currently in theduodenum. In some embodiments, the ingestible device 100 may also storea timestamp indicating the time when ingestible device 100 entered theduodenum. Once ingestible device 100 reaches the duodenum, process 500proceeds to 520 where ingestible device 100 may be configured to gatherdata in order to detect muscle contractions and detect entry into thejejunum (e.g., jejunum 314 (FIG. 3)). Process 500 also proceeds from 514to 516, where ingestible device 100 may be configured to gather dataadditional data in order to detect re-entry into the stomach (e.g.,stomach 306 (FIG. 3)) from the duodenum (e.g., duodenum 310 (FIG. 3)).

At 516, the ingestible device (e.g., ingestible device 100, 300, or 400)gathers measurements (e.g., via sensing sub-unit 126 (FIG. 2)) of greenand blue reflectance levels while in the duodenum (e.g., duodenum 310(FIG. 3)). For example, ingestible device 100 may be configured toperiodically gather measurements (e.g., via sensing sub-unit 126 (FIG.2)) of green and blue reflectance levels while in the duodenum, similarto the measurements made at 510 while in the stomach. For instance,ingestible device 100 may be configured to transmit a green illuminationand a blue illumination (e.g., via illuminator 124 (FIG. 2)) every fiveto fifteen seconds, and measure the resulting reflectance (e.g., viadetector 122 (FIG. 2)). Every time that ingestible device 100 gathers anew set of measurements, the measurements may be added to a stored dataset (e.g., stored within memory circuitry of PCB 120 (FIG. 2)). Theingestible device 100 may then use this data set to determine whether ornot ingestible device 100 is still within the duodenum (e.g., duodenum310 (FIG. 3)), or if the ingestible device 100 has transitioned backinto the stomach (e.g., stomach 306 (FIG. 3)).

At 518, the ingestible device (e.g., ingestible device 100, 300, or 400)determines a transition from the duodenum (e.g., duodenum 310 (FIG. 3))to the stomach (e.g., stomach 306 (FIG. 3)) based on a ratio of themeasured green reflectance levels to the measured blue reflectancelevels. In some embodiments, ingestible device 100 may compare the ratioof the measured green reflectance levels to the measured bluereflectance levels recently gathered by ingestible device 100 (e.g.,measurements gathered at 516), and determine whether or not the ratio ofthe measured green reflectance levels to the measured blue reflectancelevels is similar to the average ratio of the measured green reflectancelevels to the measured blue reflectance levels seen in the stomach(e.g., stomach 306 (FIG. 3)). For instance, ingestible device 100 mayretrieve data (e.g., from memory circuitry of PCB 120 (FIG. 2))indicative of the average ratio of the measured green reflectance levelsto the measured blue reflectance levels seen in the stomach, anddetermine that ingestible device 100 has transitioned back to thestomach if the recently measured ratio of the measured green reflectancelevels to the measured blue reflectance levels is sufficiently similarto the average level in the stomach (e.g., within 20% of the averageratio of the measured green reflectance levels to the measured bluereflectance levels seen in the stomach, or within any other suitablethreshold level). If the ingestible device detects a transition from theduodenum (e.g., duodenum 310 (FIG. 3)) to the stomach (e.g., stomach 306(FIG. 3)), process 500 proceeds to 508 to store data indicating theingestible device has entered the stomach (e.g., stomach 306 (FIG. 3)),and continues to monitor for further transitions. Alternatively, if theingestible device does not detect a transition from the duodenum (e.g.,duodenum 310 (FIG. 3)) to the stomach (e.g., stomach 306 (FIG. 3)),process 500 proceeds to 516 to gather additional measurements of greenand blue reflectance levels while in the duodenum (e.g., duodenum 310(FIG. 3)), which may be used to continuously monitor for possibletransitions back into the stomach. An example procedure for usingmeasurements of green and blue reflectances to monitor for transitionsbetween the stomach and the duodenum is discussed in greater detail inrelation to FIG. 6.

At 520, the ingestible device (e.g., ingestible device 100, 300, or 400)gathers periodic measurements of the reflectance levels (e.g., viasensing sub-unit 126 (FIG. 2)) while in the duodenum (e.g., duodenum 310(FIG. 3)). In some embodiments, the ingestible device (e.g., ingestibledevice 100, 300, or 400) may gather similar periodic measurements whilein the stomach as well. In some embodiments, these periodic measurementsmay enable ingestible device 100 to detect muscle contractions (e.g.,muscle contractions due to a peristaltic wave as discussed in relationto FIG. 4), which may be indicative of entry into a jejunum (e.g.,jejunum 314 (FIG. 3)). Ingestible device 100 may be configured to gatherperiodic measurements using any suitable wavelength of illumination(e.g., by generating illumination using illuminator 124, and detectingthe resulting reflectance using detector 122 (FIG. 2)), or combinationsof wavelengths of illumination. For example, in some embodiments,ingestible device 100 may be configured to generate red, green, and blueillumination, store separate data sets indicative of red, green, andblue illumination, and analyze each of the data sets separately tosearch for frequency components in the recorded data indicative ofdetected muscle contractions. In some embodiments, the measurementsgathered by ingestible device 100 at 520 may be sufficiently fast as todetect peristaltic waves in a subject. For instance, in a healthy humansubject, peristaltic waves may occur at a rate of approximately 0.1 Hzto 0.2 Hz. Therefore, the ingestible device 400 may be configured togenerate illumination and measure the resulting reflectance at leastonce every 2.5 seconds (i.e., the minimum rate necessary to detect a 0.2Hz signal), and preferably at a higher rate, such as once every 0.5seconds or faster, and store values indicative of the resultingreflectances in a data set (e.g., within memory circuitry of PCB 120(FIG. 2)). After gathering additional data (e.g., after gathering onenew data point, or a predetermined number of new data points), process500 proceeds to 522, where ingestible device 100 determines whether ornot a muscle contraction has been detected.

At 522, the ingestible device (e.g., ingestible device 100, 300, or 400)determines (e.g., via control circuitry within PCB 120 (FIG. 0.2))whether the ingestible device detects a muscle contraction based on themeasurements of reflectance levels (e.g., as gathered by sensingsub-unit 126 (FIG. 2)). For example, ingestible device 100 may obtain afixed amount of data stored as a result of measurements made at 520(e.g., retrieve the past minute of data from memory circuitry within PCB120 (FIG. 2)). Ingestible device 100 may then convert the obtained datainto the frequency domain, and search for peaks in a frequency rangethat would be consistent with peristaltic waves. For example, in ahealthy human subject, peristaltic waves may occur at a rate ofapproximately 0.1 Hz to 0.2 Hz, and an ingestible device 100 may beconfigured to search for peaks in the frequency domain representation ofthe data between 0.1 Hz and 0.2 Hz above a threshold value. If theingestible device 100 detects a contraction based on the reflectancelevels (e.g., based on detecting peaks in the frequency domainrepresentation of the data between 0.1 Hz and 0.2 Hz), process 500proceeds to 524 to store data indicating that the device has entered thejejunum. Alternatively, if the ingestible device 100 does not detect amuscle contraction, process 500 proceeds to 520 to gather periodicmeasurements of the reflectance levels while in the duodenum (e.g.,duodenum 310 (FIG. 3)). In some embodiments, the ingestible device(e.g., ingestible device 100, 300, or 400) may store data (e.g., withinmemory circuitry of PCB 120 (FIG. 2)) indicating that a musclecontraction was detected, and process 500 will not proceed from 522 to524 until a sufficient number of muscle contractions have been detected.

At 524, the ingestible device (e.g., ingestible device 100, 300, or 400)stores data (e.g., within memory circuitry of PCB 120 (FIG. 2))indicating that the device has entered the jejunum (e.g., jejunum 314(FIG. 3)). For example, in response to detecting that muscle contractionhas occurred at 522, ingestible device 100 may determine that it hasentered the jejunum 314, and is no longer inside of the duodenum (e.g.,duodenum 310 (FIG. 3)) or the stomach (e.g., stomach 306 (FIG. 3)). Insome embodiments, the ingestible device 100 may continue to measuremuscle contractions while in the jejunum, and may store data indicativeof the frequency, number, or strength of the muscle contractions overtime (e.g., within memory circuitry of PCB 120 (FIG. 2)). In someembodiments, the ingestible device 100 may also be configured to monitorfor one or more transitions. Such transitions can include a transitionfrom the jejunum to the ileum, an ileoceacal transition from the ileumto the cecum, a transition from the cecum to the colon, or detect exitfrom the body (e.g., by measuring reflectances, temperature, or levelsof ambient light).

In some embodiments, the ingestible device (e.g., ingestible device 100,300, or 400) may also determine that it has entered the jejunum (e.g.,jejunum 314 (FIG. 3)) after a pre-determined amount of time has passedafter having detected entry into the duodenum (e.g., duodenum 310 (FIG.3)). For example, barring a reverse pyloric transition from the duodenum(e.g., duodenum 310 (FIG. 3)) back to the stomach (e.g., stomach 306(FIG. 3)), the typical transit time for an ingestible device to reachthe jejunum from the duodenum in a healthy human subject is less thanthree minutes. In some embodiments, the ingestible device (e.g.,ingestible device 100, 300, or 400) may therefore be configured toautomatically determine that it has entered the jejunum after spendingat least three minutes within the duodenum. This determination may bemade separately from the determination made based on measured musclecontractions (e.g., the determination made at 522), and in someembodiments, ingestible device 100 may determine that it has entered thejejunum in response to either detecting muscle contractions, or afterthree minutes has elapsed from having entered the duodenum (e.g., asdetermined by storing data at 514 indicative of the time that ingestibledevice entered the duodenum).

For illustrative purposes, 512-518 of process 500 describe theingestible device (e.g., ingestible device 100, 300, or 400) measuringgreen reflectances and blue reflectances, calculating a ratio of the tworeflectances, and using this information to determine when theingestible device has transitioned between the duodenum and stomach.However, in some embodiments, other wavelengths of light may be usedother than green and blue, provided that the wavelengths of light chosenhave different reflective properties within the stomach and the duodenum(e.g., as a result of different reflection coefficients of the stomachtissue and the tissue of the duodenum).

It will be understood that the steps and descriptions of the flowchartsof this disclosure, including FIG. 5, are merely illustrative. Any ofthe steps and descriptions of the flowcharts, including FIG. 5, may bemodified, omitted, rearranged, and performed in alternate orders or inparallel, two or more of the steps may be combined, or any additionalsteps may be added, without departing from the scope of the presentdisclosure. For example, the ingestible device 100 may calculate themean and the standard deviation of multiple data sets in parallel inorder to speed up the overall computation time. As another example,ingestible device 100 may gather data periodic measurements and detectpossible muscle contractions (e.g., at 520-522) while simultaneouslygathering green and blue reflectance levels to determine transitions toand from the stomach and duodenum (e.g., at 510-518). Furthermore, itshould be noted that the steps and descriptions of FIG. 5 may becombined with any other system, device, or method described in thisapplication, including processes 600 (FIG. 6) and 900 (FIG. 9), and anyof the ingestible devices or systems discussed in this application(e.g., ingestible devices 100, 300, or 400) could be used to perform oneor more of the steps in FIG. 5.

FIG. 6 is a flowchart illustrating some aspects of a process fordetecting transitions from a stomach to a duodenum and from a duodenumback to a stomach, which may be used when determining a location of aningestible device as it transits through a gastrointestinal (GI) tract,in accordance with some embodiments of the disclosure. In someembodiments, process 600 may begin when an ingestible device firstdetects that it has entered the stomach, and will continue as long asthe ingestible device determines that it is within the stomach or theduodenum. In some embodiments, process 600 may only be terminated whenan ingestible device determines that it has entered the jejunum, orotherwise progressed past the duodenum and the stomach. Although FIG. 6may be described in connection with the ingestible device 100 forillustrative purposes, this is not intended to be limiting, and eitherportions or the entirety of the duodenum detection process 600 describedin FIG. 6 may be applied to any device discussed in this application(e.g., the ingestible devices 100, 300, or 400), and any of theingestible devices may be used to perform one or more parts of theprocess described in FIG. 6. Furthermore, the features of FIG. 6 may becombined with any other systems, methods or processes described in thisapplication. For example, portions of the process described by theprocess in FIG. 6 may be integrated into process 500 discussed inrelation to FIG. 5.

At 602, the ingestible device (e.g., ingestible device 100, 300, or 400)retrieves a data set (e.g., from memory circuitry within PCB 120 (FIG.2)) with ratios of the measured green reflectance levels to the measuredblue reflectance levels over time. For example, ingestible device 100may retrieve a data set from PCB 120 containing recently recorded ratiosof the measured green reflectance levels to the measured bluereflectance levels (e.g., as recorded at 510 or 516 of process 500 (FIG.5)). In some embodiments, the retrieved data set may include the ratiosof the measured green reflectance levels to the measured bluereflectance levels over time. Example plots of data sets of ratios ofthe measured green reflectance levels to the measured blue reflectancelevels are discussed further in relation to FIG. 7 and FIG. 8.

At 604, the ingestible device (e.g., ingestible device 100, 300, or 400)includes a new measurement (e.g., as made with sensing sub-unit 126(FIG. 2)) of a ratio of the measured green reflectance level to themeasured blue reflectance level in the data set. For example, ingestibledevice 100 may be configured to occasionally record new data bytransmitting green and blue illumination (e.g., via illuminator 124(FIG. 2)), detecting the amount of reflectance received due to the greenand blue illumination (e.g., via detector 122 (FIG. 2)), and storingdata indicative of the amount of the received reflectance (e.g., inmemory circuitry of PCB 120 (FIG. 2)). The ingestible device 100 may beconfigured to record new data every five to fifteen seconds, or at anyother convenient interval of time. For illustrative purposes, ingestibledevice 100 is described as storing and retrieving the ratio of themeasured green reflectance levels to the measured blue reflectancelevels (e.g., if the amount of detected green reflectance was identicalto the amount of detected blue reflectance at a given time, the ratio ofthe green and blue reflectances would be “1.0” at that given time);however, it is understood that the green reflectance data and the bluereflectance data may be stored separately within the memory ofingestible device 100 (e.g., stored as two separate data sets withinmemory circuitry of PCB 120 (FIG. 2)).

At 606, the ingestible device (e.g., ingestible device 100, 300, or 400)retrieves a first subset of recent data by applying a first slidingwindow filter to the data set. For example, ingestible device 100 mayuse a sliding window filter to obtain a predetermined amount of the mostrecent data within the data set, which may include any new values of theratio of the measured green reflectance level to the measured bluereflectance level obtained at 604. For instance, the ingestible devicemay be configured to select between ten and forty data points from thedata set, or ingestible device 100 may be configured to select apredetermined range of data values between fifteen seconds of data andfive minutes of data. In some embodiments, other ranges of data may beselected, depending on how frequently measurements are recorded, and theparticular application at hand. For instance, any suitable amount ofdata may be selected in the sliding window, provided that it issufficient to detect statistically significant differences between thedata selected in a second sliding window (e.g., the second subset ofdata selected at 614).

In some embodiments, the ingestible device (e.g., ingestible device 100,300, or 400) may also be configured to remove outliers from the dataset, or to smooth out unwanted noise in the data set. For example,ingestible device 100 may select the first subset of data, or any othersubset of data, by first obtaining a raw set of values by applying awindow filter to the data set (e.g., selecting a particular range ofdata to be included). Ingestible device 100 may then be configured toidentify outliers in the raw set of values; for instance, by identifyingdata points that are over three standard deviations away from the meanvalue of the raw set of values, or any other suitable threshold.Ingestible device 100 may then determine the subset of data by removingoutliers from the raw set of values. This may enable ingestible device100 to avoid spurious information when determining whether or not it islocated within the stomach or the duodenum.

At 608, the ingestible device (e.g., ingestible device 100, 300, or 400)determines whether the most recently detected location was the duodenum(e.g., duodenum 310 (FIG. 3)). In some embodiments, ingestible device100 may store a data flag (e.g., within memory circuitry of PCB 120(FIG. 2)) indicating the most recent portion of the GI tract that theingestible device 100 detected itself to be within. For instance, everytime ingestible device 100 detects entry to the stomach (e.g., detectsentry into stomach 306 (FIG. 3) as a result of the decision made at610), a flag is stored in memory indicating the ingestible device 100 isin the stomach (e.g., as part of storing data at 612). If ingestibledevice 100 subsequently detects entry into the duodenum (e.g., detectsentry into duodenum 310 (FIG. 3) as a result of a decision made at 624),another different flag is stored in memory indicating that theingestible device 100 is in the duodenum (e.g., as part of storing dataat 624). In this case, ingestible device 100 may retrieve the mostrecently stored flag at 608, and determine whether or not the flagindicates that the ingestible device 100 was most recently within theduodenum. If ingestible device 100 detects that it was most recently inthe duodenum, process 600 proceeds to 610 where the ingestible devicecompares the recent measurements of the ratios of the measured greenreflectance levels to the measured blue reflectance levels (e.g.,measurements that include the recent measurement made at 606) to thetypical ratios measured within the stomach, and uses this information todetermine whether a reverse pyloric transition from the duodenum back tothe stomach has occurred. Alternately, if ingestible device 100 detectsthat it was not most recently in the duodenum (e.g., because it was inthe stomach instead), process 600 proceeds to 614 where the ingestibledevice compares the recent measurements of the ratios of the measuredgreen reflectance levels to the measured blue reflectance levels (e.g.,measurements that include the recent measurement made at 606) to pastmeasurements, and uses this information to determine whether a pylorictransition from the stomach to the duodenum has occurred.

Process 600 proceeds from 608 to 610 when the ingestible devicedetermined that it was most recently in the duodenum. At 610, theingestible device (e.g., ingestible device 100, 300, or 400) determines(e.g., via control circuitry within PCB 120 (FIG. 2)) whether thecurrent G/B signal is similar to a recorded average G/B signal in thestomach. For example, ingestible device 100 may be configured to havepreviously stored data (e.g., within memory circuitry of PCB 120 (FIG.2)) indicative of the average ratio of the measured green reflectancelevels to the measured blue reflectance levels measured in the stomach.Ingestible device 100 may then retrieve this stored data indicative ofthe average ratio of the measured green reflectance levels to themeasured blue reflectance levels in the stomach, and compare thisagainst the recent measurements in order to determine whether or notingestible device 100 has returned back to the stomach from theduodenum. For instance, ingestible device 100 may determine if the meanvalue of the first subset of recent data (i.e., the average value of therecently measured ratios of the measured green reflectance levels to themeasured blue reflectance levels) is less than the average ratio of themeasured green reflectance levels to the measured blue reflectancelevels within the stomach, or less that the average ratio measuredwithin the stomach plus a predetermined number times the standarddeviation of the ratios measured within the stomach. For instance, ifthe average ratio of the measured green reflectance levels to themeasured blue reflectance levels in the stomach was “1,” with a standarddeviation of “0.2,” ingestible device 100 may determine whether or notthe mean value of the first subset of data is less than “1.0+k*0.2,”where “k” is a number between zero and five. It is understood that, insome embodiments, the ingestible device 100 may be configured to use adifferent threshold level to determine whether or not the mean value ofthe first subset of recent data is sufficiently similar to the averageratio of the measured green reflectance levels to the measured bluereflectance levels within the stomach. In response to determining thatthe recent ratio of the measured green reflectance levels to themeasured blue reflectance levels is similar to the average ratio ofmeasured green and blue reflectance levels seen in the stomach, process600 proceeds to 612 where ingestible device 100 stores data indicatingthat it has re-entered the stomach from the duodenum. Alternately, inresponse to determining that the recent ratio of measured green and bluereflectance levels is sufficiently different from the average ratio ofmeasured green and blue reflectance levels seen in the stomach,ingestible device 100 proceeds directly to 604, and continues to obtainnew data on an ongoing basis.

At 612, the ingestible device (e.g., ingestible device 100, 300, or 400)stores data indicating a reverse pyloric transition from the duodenum tothe stomach was detected. For example, ingestible device 100 may store adata flag (e.g., within memory circuitry of PCB 120 (FIG. 2)) indicatingthat the ingestible device 100 most recently detected itself to bewithin the stomach portion of the GI tract (e.g., stomach 306 (FIG. 3)).In some embodiments, ingestible device 100 may also store data (e.g.,within memory circuitry of PCB 120 (FIG. 2)) indicating a time thatingestible device 100 detected the reverse pyloric transition from theduodenum to the stomach. This information may be used by ingestibledevice 100 at 608, and as a result process 600 may proceed from 608 to614, rather than proceeding from 618 to 610. After ingestible device 100stores the data indicating a reverse pyloric transition from theduodenum to the stomach was detected, process 600 proceeds to 604 whereingestible device 100 continues to gather additional measurements, andcontinues to monitor for further transitions between the stomach and theduodenum.

Process 600 proceeds from 608 to 614 when the ingestible devicedetermined that it was not most recently in the duodenum (e.g., as aresult of having most recently been in the stomach instead). At 614, theingestible device (e.g., ingestible device 100, 300, or 400) retrieves asecond subset of previous data by applying a second sliding windowfilter to the data set. For example, ingestible device 100 may use asliding window filter to obtain a predetermined amount of older datafrom a past time range, which may be separated from recent time rangeused to select the first subset of data gathered at 606 by apredetermined period of time. In some embodiments, any suitable amountof data may be selected by the first and second window filters, and thefirst and second window filters may be separated by any appropriatepredetermined amount of time. For example, in some embodiments, thefirst window filter and the second window filter may each be configuredto select a predetermined range of data values from the data set, thepredetermined range being between fifteen seconds of data and fiveminutes of data. In some embodiments, the recent measurements and thepast measurements may then be separated by a predetermined period oftime that is between one to five times the predetermined range of datavalues. For instance, ingestible device 100 may select the first subsetof data and the second subset of data to each be one minute of dataselected from the dataset (i.e., selected to have a predetermined rangeof one minute), and the first subset of data and the second subset ofdata are selected from recorded measurements that are at least twominutes apart (i.e., the predetermined period of time is two minutes,which is twice the range used to select the subsets of data using thewindow filters). As another example, ingestible device 100 may selectthe first subset of data and the second subset of data to each be fiveminutes of data selected from the dataset (i.e., selected to have apredetermined range of five minutes), and the first subset of data andthe second subset of data are selected from recorded measurements thatare at least 10 minutes apart (i.e., the predetermined period of time istwo minutes, which is twice the range used to select the subsets of datausing the window filters).

In some embodiments, if ingestible device 100 recently transitioned tothe stomach from the duodenum (e.g., as determined by checking forrecent data stored within ingestible device 100 at 612), ingestibledevice 100 may select the second subset of data at 614 from a time framewhen ingestible device 100 is known to be within the stomach. In someembodiments, ingestible device 100 may alternately select a previouslyrecorded average and standard deviation for ratios of green reflectancesand blue reflectances within the stomach (e.g., an average and standarddeviation typical of data recorded within the stomach, as previouslyrecorded within memory circuitry of PCB 120 at 620) in place of thesecond subset of data. In this case, ingestible device 100 may simplyuse the previously recorded average and previously recorded standarddeviation when making a determination at 616, rather than expendingresources to calculate the mean and standard deviation of the secondsubset.

At 616, the ingestible device (e.g., ingestible device 100, 300, or 400)determines whether the difference between the mean of the second subsetand the mean of the first subset is greater than a predeterminedmultiple of the standard deviation of the first subset. For example,ingestible device 100 may compute a difference between a mean of thefirst subset of recent data and a mean of a second subset of past data,and determine whether this difference is greater than three times thestandard deviation of the second subset of past data. In someembodiments, it is understood that any convenient threshold level may beused other than three times the standard deviation, such as any valuebetween one and five times the standard deviation. Also, in someembodiments, the ingestible device may instead set the threshold levelbased on the standard deviation of the second subset instead of thefirst subset. In response to determining that the difference between themean of the first subset and the mean of the second subset is greaterthan a predetermined multiple of the standard deviation of the secondsubset, process 600 proceeds to 618. Otherwise, process 600 proceedsback to 604, where the ingestible device 604 continues to gather newdata to be used in monitoring for transitions between the stomach (e.g.,stomach 306 (FIG. 3)) and the duodenum (e.g., duodenum 310 (FIG. 3)).

At 618, the ingestible device (e.g., ingestible device 100, 300, or 400)determines (e.g., via control circuitry within PCB 120 (FIG. 2)) whetherthe determination made at 616 is the first time that the differencebetween the mean of the first subset of recent data and the mean of thesecond subset of past data is calculated to be greater than the standarddeviation of the second subset. If the ingestible device determines thatthis is the first time that the difference between the mean of the firstsubset and the mean of the second subset is calculated to be greaterthan the standard deviation of the second subset, process 600 proceedsto 620 to store the mean of the second subset of past data as an averageG/B signal in the stomach. Alternatively, if the ingestible devicedetermines that the immediately preceding determination made at 616 isnot the first time that the difference between the mean of the firstsubset of recent data and the mean of the second subset of past data iscalculated to be greater than the standard deviation of the secondsubset, process 600 proceeds directly to 622.

At 620, the ingestible device (e.g., ingestible device 100, 300, or 400)stores the mean of the second subset as an average G/B signal in thestomach. For example, ingestible device 100 may be configured to storethe mean of the second subset of past data (e.g., store within memorycircuitry of PCB 120 (FIG. 2)) as the average ratio of the measuredgreen reflectance levels to the measured blue reflectance levelsmeasured in the stomach. In some embodiments, ingestible device 100 mayalso store the standard deviation of the second subset of past data as atypical standard deviation of the ratios of the measured greenreflectance levels to the measured blue reflectance levels detectedwithin the stomach. This stored information may be used by theingestible device later on (e.g., at 610) to compare against futuredata, which may enable the ingestible device to detect reverse pylorictransitions from the duodenum (e.g., duodenum 310 (FIG. 3)) back to thestomach (e.g., stomach 306 (FIG. 3)), and may generally be used in placeof other experimental data gathered from the stomach (e.g., in place ofthe second subset of data at 616). After storing the mean of the secondsubset as an average G/B signal in the stomach, process 600 proceeds to622.

At 622, the ingestible device (e.g., ingestible device 100, 300, or 400)determines whether a difference of the mean of the first subset ofrecent data to the mean of the second subset of past data is greaterthan a predetermined threshold, “M”. In some embodiments, thepredetermined threshold, “M,” will be sufficiently large to ensure thatthe mean of the first subset is substantially larger than the mean ofthe second subset, and may enable ingestible device 100 to ensure thatit detected an actual transition to the duodenum. This may beparticularly advantageous when the determination made at 616 ispotentially unreliable due to the standard deviation of the secondsubset of past data being abnormally small. For example, a typical valueof the predetermined threshold “M,” may be on the order of 0.1 to 0.5.If ingestible device 100 determines that the difference of the mean ofthe first subset of recent data to the second subset of past data isgreater than a predetermined threshold, process 600 proceeds to 624 tostore data indicating that a pyloric transition from the stomach to theduodenum (e.g., from stomach 306 to duodenum 310 (FIG. 3)) was detected.Alternatively, if the ingestible device determines that the ratio of themean of the first subset to the second subset is less than or equal tothe predetermined threshold, “M” (i.e., determines that a transition tothe duodenum has not occurred), process 600 proceeds directly to 604where ingestible device 100 continues to make new measurements andmonitor for possible transitions between the stomach and the duodenum.

In some embodiments, instead of using a difference of the mean of thefirst subset of recent data to the mean of the second subset of pastdata, the ingestible device (e.g., ingestible device 100, 300, or 400)determines whether the ratio of the mean of the first subset of recentdata to the mean of the second subset of past data is greater than apredetermined threshold, “M”. In some embodiments, the predeterminedthreshold, “M,” will be sufficiently large to ensure that the mean ofthe first subset is substantially larger than the mean of the secondsubset, and may enable ingestible device 100 to ensure that it detectedan actual transition to the duodenum. This may be particularlyadvantageous when the determination made at 616 is potentiallyunreliable due to the standard deviation of the second subset of pastdata being abnormally small. For example, a typical value of thepredetermined threshold “M,” may be on the order of 1.2 to 2.0. It isunderstood any convenient type of threshold or calculation may be usedto determine whether or not the first subset of data and the secondsubset of data are both statistically distinct from one another, andalso substantially different from one another in terms of overallaverage value.

At 624, the ingestible device (e.g., ingestible device 100, 300, or 400)stores data indicating a pyloric transition from the stomach to theduodenum was detected. For example, ingestible device 100 may store adata flag (e.g., within memory circuitry of PCB 120 (FIG. 2)) indicatingthat the ingestible device 100 most recently detected itself to bewithin the duodenum portion of the GI tract (e.g., duodenum 310 (FIG.3)). In some embodiments, ingestible device 100 may also store data(e.g., within memory circuitry of PCB 120 (FIG. 2)) indicating a timethat ingestible device 100 detected the pyloric transition from thestomach to the duodenum. This information may be used by ingestibledevice 100 at 608, and as a result process 600 may proceed from 608 to610, rather than proceeding from 618 to 614. After ingestible device 100stores the data indicating a pyloric transition from the stomach to theduodenum was detected, process 600 proceeds to 604 where ingestibledevice 100 continues to gather additional measurements, and continues tomonitor for further transitions between the stomach and the duodenum.

It will be understood that the steps and descriptions of the flowchartsof this disclosure, including FIG. 6, are merely illustrative. Any ofthe steps and descriptions of the flowcharts, including FIG. 6, may bemodified, omitted, rearranged, and performed in alternate orders or inparallel, two or more of the steps may be combined, or any additionalsteps may be added, without departing from the scope of the presentdisclosure. For example, the ingestible device 100 may calculate themean and the standard deviation of multiple data sets in parallel inorder to speed up the overall computation time. Furthermore, it shouldbe noted that the steps and descriptions of FIG. 6 may be combined withany other system, device, or method described in this application, andany of the ingestible devices or systems discussed in this applicationcould be used to perform one or more of the steps in FIG. 6. Forexample, portions of process 600 may be incorporated into 508-516 ofprocess 500 (FIG. 5), and may be part of a more general process fordetermining a location of the ingestible device. As another example, theratio of detected blue and green light (e.g., as measured and added tothe data set at 604) may continue even outside of the stomach orduodenum, and similar information may be recorded by the ingestibledevice throughout its transit in the GI tract. Example plots of datasets of ratios of measured green and blue reflectance levels, which maybe gathered throughout the GI tract, are discussed further in relationto FIG. 7 and FIG. 8 below.

FIG. 7 is a plot illustrating data collected during an example operationof an ingestible device (e.g., ingestible device 100, 300, or 400),which may be used when determining a location of an ingestible device asit transits through a gastrointestinal (GI) tract, in accordance withsome embodiments of the disclosure.

Although FIG. 7 may be described in connection with ingestible device100 for illustrative purposes, this is not intended to be limiting, andplot 700 and data set 702 may be typical of data gathered by any devicediscussed in this application. Plot 700 depicts the ratios of themeasured green reflectance levels to the measured blue reflectancelevels over time. For example, ingestible device 100 may have computedthe value for each point in the data set 702 by transmitting green andblue illumination at a given time (e.g., via illuminator 124 (FIG. 2)),measuring the resulting green and blue reflectances (e.g., via detector122 (FIG. 2)), calculating the ratio of the resulting reflectances, andstoring the ratio in the data set along with a timestamp indicating thetime that the reflectances were gathered.

At 704, shortly after ingestible device 100 begins operation, ingestibledevice 100 determines that it has reached at least the stomach (e.g., asa result of making a determination similar to the determinationdiscussed in relation to 506 in process 500 (FIG. 5)). Ingestible device100 continues to gather additional measurements of green and bluereflectance levels, and at 706 ingestible device 100 determines that apyloric transition has occurred from the stomach to the duodenum (e.g.,as a result of making a determination similar to the determinationsdiscussed in relation to 616-624 of process 600 (FIG. 6)). Notably, thevalues in data set 702 around 706 jump up precipitously, which isindicative of the higher ratios of measured green reflectance levels tomeasured blue reflectance levels typical of the duodenum.

The remainder of the data set 702 depicts the ratios of the measuredgreen reflectance levels to the measured blue reflectance levelsthroughout the remainder of the GI tract. At 708, ingestible device 100has reached the jejunum (e.g., as determined through measurements ofmuscle contractions, as discussed in relation to FIG. 9), and by 710,ingestible device 100 has reached the cecum. It is understood that, insome embodiments, the overall character and appearance of data set 702changes within the small intestine (i.e., the duodenum, jejunum, andileum) versus the cecum. Within the jejunum and ileum, there maytypically be a wide variation in the ratios of the measured greenreflectance levels to the measured blue reflectance levels, resulting inrelatively noisy data with a high standard deviation. By comparison,within the cecum ingestible device 100 may measure a relatively stableratio of the measured green reflectance levels to the measured bluereflectance levels. In some embodiments, ingestible device 100 may beconfigured to determine transitions from the small intestine to thececum based on these differences. For example, ingestible device 100 maycompare recent windows of data to past windows of data, and detect atransition to the cecum in response to determining that the standarddeviation of the ratios in the recent window of data is substantiallyless than the standard deviation of the ratios in the past window ofdata.

FIG. 8 is another plot illustrating data collected during an exampleoperation of an ingestible device, which may be used when determining alocation of an ingestible device as it transits through agastrointestinal (GI) tract, in accordance with some embodiments of thedisclosure. Similar to FIG. 7, FIG. 8 may be described in connectionwith the ingestible device 100 for illustrative purposes. However, thisis not intended to be limiting, and plot 800 and data set 802 may betypical of data gathered by any device discussed in this application.

At 804, shortly after ingestible device 100 begins operation, ingestibledevice 100 determines that it has reached at least the stomach (e.g., asa result of making a determination similar to the determinationdiscussed in relation to 506 in process 500 (FIG. 5)). Ingestible device100 continues to gather additional measurements of green and bluereflectance levels (e.g., via sensing sub-unit 126 (FIG. 2)), and at 806ingestible devices 100 determines that a pyloric transition has occurredfrom the stomach to the duodenum (e.g., as a result of making adetermination similar to the determinations discussed in relation to616-624 of process 600 (FIG. 6)). Notably, the values in data set 802around 806 jump up precipitously, which is indicative of the higherratios of measured green reflectance levels to measured blue reflectancelevels typical of the duodenum, before falling shortly thereafter. As aresult of the reduced values in data set 802, ingestible device 100determines that a reverse pyloric transition has occurred from theduodenum back to the stomach at 808 (e.g., as a result of making adetermination similar to the determinations discussed in relation to610-612 of process 600 (FIG. 6)). At 810, as a result of the values indata set 802 increasing again, ingestible device 100 determines thatanother pyloric transition has occurred from the stomach to theduodenum, and shortly thereafter ingestible device 100 proceeds onwardsto the jejunum, ileum, and cecum.

The remainder of the data set 802 depicts the ratios of the measuredgreen reflectance levels to the measured blue reflectance levelsthroughout the remainder of the GI tract. Notably, at 812, ingestibledevice reaches the transition point between the ileum and the cecum. Asdiscussed above in relation to FIG. 7, the transition to the cecum ismarked by a reduced standard deviation in the ratios of measured greenreflectances and measured blue reflectances over time, and ingestibledevice 100 may be configured to detect a transition to the cecum basedon determining that the standard deviation of a recent set ofmeasurements is substantially smaller than the standard deviation ofpast measurements taken from the jejunum or ileum.

FIG. 9 is a flowchart of illustrative steps for detecting a transitionfrom a duodenum to a jejunum, which may be used when determining alocation of an ingestible device as it transits through agastrointestinal (GI) tract, in accordance with some embodiments of thedisclosure. Although FIG. 9 may be described in connection with theingestible device 100 for illustrative purposes, this is not intended tobe limiting, and either portions or the entirety of process 900described in FIG. 9 may be applied to any device discussed in thisapplication (e.g., the ingestible devices 100, 300, and 400), and any ofthese ingestible devices may be used to perform one or more parts of theprocess described in FIG. 9. Furthermore, the features of FIG. 9 may becombined with any other systems, methods or processes described in thisapplication. For example, portions of the process described by theprocess in FIG. 9 may be integrated into the localization processdescribed by FIG. 5 (e.g., as part of 520-524 of process 500 (FIG. 5)).In some embodiments, an ingestible device 100 may perform process 900while in the duodenum, or in response to detecting entry to theduodenum. In other embodiments, an ingestible device 100 may performprocess 900 while in the stomach, or in response to detecting entry intothe GI tract. It is also understood that process 900 may be performed inparallel with any other process described in this disclosure (e.g.,process 600 (FIG. 6)), which may enable ingestible device 100 to detectentry into various portions of the GI tract, without necessarilydetecting entry into a preceding portion of the GI tract.

For illustrative purposes, FIG. 9 may be discussed in terms ofingestible device 100 generating and making determinations based on asingle set of reflectance levels generated at a single wavelength by asingle sensing sub-unit (e.g., sensing sub-unit 126 (FIG. 2)). However,it is understood that ingestible device 100 may generate multiplewavelengths of illumination from multiple different sensing sub-unitspositioned around the circumference of ingestible device (e.g., multiplesensing sub-units positioned at different locations behind window 114 ofingestible device 100 (FIG. 1), and each of the resulting reflectancesmay be stored as a separate data set. Moreover, each of these sets ofreflectance levels may be used to detect muscle contractions by runningmultiple versions of process 900, each one of which processes data for adifferent set of reflectances corresponding to data sets obtained frommeasurements of different wavelengths or measurements made by differentsensing sub-units.

At 902, the ingestible device (e.g., ingestible device 100, 300, or 400)retrieves a set of reflectance levels. For example, ingestible device100 may retrieve a data set of previously recorded reflectance levelsfrom memory (e.g., from memory circuitry of PCB 120 (FIG. 2)). Each ofthe reflectance levels may correspond to reflectances previouslydetected by ingestible device 100 (e.g., via detector 122 (FIG. 2)) fromillumination generated by ingestible device 100 (e.g., via illuminator124 (FIG. 2)), and may represent a value indicative of an amount oflight detected in a given reflectance. However, it is understood thatany suitable frequency of light may be used, such as light in theinfrared, visible, or ultraviolet spectrums. In some embodiments, thereflectance levels may correspond to reflectances previously detected byingestible device 100 at periodic intervals.

At 904, the ingestible device (e.g., ingestible device 100, 300, or 400)includes new measurements of reflectance levels in the data set. Forexample, ingestible device 100 may be configured to detect a newreflectance (e.g., transmit illumination and detect the resultingreflectance using sensing sub-unit 126 (FIG. 2)) at regular intervals,or with sufficient speed as to detect peristaltic waves. For example,ingestible device 100 may be configured to generate illumination andmeasure the resulting reflectance once every three seconds (i.e., theminimum rate necessary to detect a 0.17 Hz signal), and preferably at ahigher rate, as fast at 0.1 second or even faster. It is understood thatthe periodic interval between measurements may be adapted as neededbased on the species of the subject, and the expected frequency of theperistaltic waves to be measured. Every time ingestible device 100 makesa new reflectance level measurement at 904, the new data is included tothe data set (e.g., a data set stored within memory circuitry of PCB 120(FIG. 2)).

At 906, the ingestible device (e.g., ingestible device 100, 300, or 400)obtains a first subset of recent data by applying a sliding windowfilter to the data set. For example, ingestible device 100 may retrievea one-minute worth of data from the data set. If the data set includesvalues for reflectances measured every second, this would beapproximately 60 data points worth of data. Any suitable type of windowsize may be used, provided that the size of the window is sufficientlylarge to detect peristaltic waves (e.g., fluctuations on the order of0.1 Hz to 0.2 Hz for healthy human subjects). In some embodiments,ingestible device 100 may also clean the data, for example, by removingoutliers from the first subset of data obtained through the use of thesliding window filter.

At 908, the ingestible device (e.g., ingestible device 100, 300, or 400)obtains a second subset of recent data by interpolating the first subsetof recent data. For example, ingestible device 100 may interpolate thefirst subset of data in order to generate a second subset of data with asufficient number of data points (e.g., data points spaced every 0.5seconds or greater). In some embodiments, this may enable ingestibledevice 100 to also replace any outlier data points that may have beenremoved as part of applying the window filter at 906.

At 910, the ingestible device (e.g., ingestible device 100, 300, or 400)calculates a normalized frequency spectrum from the second subset ofdata. For example, ingestible device 100 may be configured to perform afast Fourier transform to convert the second subset of data from a timedomain representation into a frequency domain representation. It isunderstood that depending on the application being used, and the natureof the subset of data, any number of suitable procedures (e.g., Fouriertransform procedures) may be used to determine a frequency spectrum forthe second subset of data. For example, the sampling frequency and sizeof the second subset of data may be known in advance, and ingestibledevice 100 may be configured to have pre-stored values of a normalizeddiscreet Fourier transform (DFT) matrix, or the rows of the DFT matrixcorresponding to the 0.1 Hz to 0.2 Hz frequency components of interest,within memory (e.g., memory circuitry of PCB 120 (FIG. 2)). In thiscase, the ingestible device may use matrix multiplication between theDFT matrix and the data set to generate an appropriate frequencyspectrum. An example data set and corresponding frequency spectrum thatmay be obtained by the ingestible device is discussed in greater detailin relation to FIG. 10.

At 912, the ingestible device (e.g., ingestible device 100, 300, or 400)determines whether at least a portion of the normalized frequencyspectrum is between 0.1 Hz and 0.2 Hz above a threshold value of 0.5 Hz.Peristaltic waves in a healthy human subject occur at a rate between 0.1Hz and 0.2 Hz, and an ingestible device experiencing peristaltic waves(e.g., ingestible device 400 detecting contractions in walls 406 of thejejunum (FIG. 4)) may detect sinusoidal variations in the amplitude ofdetected reflectances levels that follow a similar 0.1 Hz to 0.2 Hzfrequency. If the ingestible device determines that a portion of thenormalized frequency spectrum between 0.1 Hz and 0.2 Hz is above athreshold value of 0.5, this measurement may be consistent withperistaltic waves in a healthy human subject, and process 900 proceedsto 914 where ingestible device 100 stores data indicating a musclecontraction was detected. Alternatively, if the ingestible devicedetermines that no portion of the normalized frequency spectrum between0.1 Hz and 0.2 Hz above a threshold value of 0.5, process 900 proceedsdirectly to 904 to make new measurements and to continue to monitor fornew muscle contractions. It is understood that a threshold value otherthan 0.5 may be used, and that the exact threshold may depend on thesampling frequency and type of frequency spectrum used by ingestibledevice 100.

At 914, the ingestible device (e.g., ingestible device 100, 300, or 400)stores data indicating a muscle contraction was detected. For example,ingestible device 100 may store data in memory (e.g., memory circuitryof PCB 120 (FIG. 2)) indicating that a muscle contraction was detected,and indicating the time that the muscle contraction was detected. Insome embodiments, ingestible device 100 may also monitor the totalnumber of muscle contractions detected, or the number of musclecontractions detected in a given time frame. In some embodiments,detecting a particular number of muscle contractions may be consistentwith ingestible device 100 being within the jejunum (e.g., jejunum 314(FIG. 3)) of a healthy human subject. After detecting a musclecontraction, process 900 proceeds to 916.

At 916, the ingestible device (e.g., ingestible device 100, 300, or 400)determines whether a total number of muscle contractions exceeds apredetermined threshold number. For example, ingestible device 100 mayretrieve the total number of muscle contractions detected from memory(e.g., from memory circuitry of PCB 120 (FIG. 2)), and compare the totalnumber to a threshold value. In some embodiments, the threshold valuemay be one, or any number larger than one. The larger the thresholdvalue, the more muscle contractions need to be detected beforeingestible device 100 stores data indicating that it has entered thejejunum. In practice, setting the threshold value as three or higher mayprevent the ingestible device from detecting false positives (e.g., dueto natural movement of the GI tract organs, or due to movement of thesubject). If the total number of contractions exceeds the predeterminedthreshold number, process 900 proceeds to 918 to store data indicatingdetection of a transition from the duodenum to the jejunum.Alternatively, if the total number of contractions does not exceed apredetermined threshold number, process 900 proceeds to 904 to includenew measurements of reflectance levels in the data set. An example plotof the muscle contractions detected over time is discussed in greaterdetail in relation to FIG. 11.

At 918, the ingestible device (e.g., ingestible device 100, 300, or 400)stores data indicating detection of a transition from the duodenum tothe jejunum. For example, ingestible device 100 may store data in memory(e.g., from memory circuitry of PCB 120 (FIG. 2)) indicating that thejejunum has been reached. In some embodiments, if ingestible device 100is configured to perform all or part of process 900 while in thestomach, ingestible device 100 may store data at 918 indicatingdetection of a transition from the stomach directly to the jejunum(e.g., as a result of transitioning too quickly through the duodenum forthe pyloric transition to be detected using process 600 (FIG. 6)).

In some embodiments, the ingestible device (e.g., ingestible device 100,300, or 400) may be configured to obtain a fluid sample from theenvironment external to a housing of the ingestible device in responseto identifying a change in the location of the ingestible device.

For example, ingestible device 100 may be configured to obtain a fluidsample from the environment external to the housing of ingestible device100 (e.g., through the use of optional opening 116 and optional rotatingassembly 118 (FIG. 2)) in response to determining that the ingestibledevice is located within the jejunum (e.g., jejunum 314 (FIG. 3)). Insome embodiments, ingestible device 100 may also be equipped withappropriate diagnostics to detect certain medical conditions based onthe retrieved fluid sample, such as small intestinal bacterialovergrowth (SIBO).

In some embodiments, the ingestible device (e.g., ingestible device 100,300, or 400) may be configured to deliver a dispensable substance thatis pre-stored within the ingestible device from the ingestible deviceinto the gastrointestinal tract in response to identifying the change inthe location of the ingestible device. For example, ingestible device100 may have a dispensable substance pre-stored within the ingestibledevice 100 (e.g., within a storage chamber or cavity on optional storagesub-unit 118-3 (FIG. 2)), and ingestible device 100 may be configured todispense the substance into the gastrointestinal tract (e.g., throughthe use of optional opening 116 and optional rotating assembly 118 (FIG.2)) when the ingestible device 100 detects that the ingestible device100 is located within the jejunum (e.g., jejunum 314 (FIG. 3)). In someembodiments, this may enable ingestible device 100 to deliver substances(e.g., therapeutics and medicaments) at targeted locations within the GItract.

In some embodiments, the ingestible device (e.g., ingestible device 100,300, or 400) may be configured to perform an action based on the totalnumber of detected muscle contractions. For example, ingestible device100 may be configured to retrieve data indicative of the total number ofmuscle contractions (e.g., from memory circuitry of PCB 120 (FIG. 2)),and compare that to an expected number of muscle contractions in ahealthy individual. In response, the ingestible device may eitherdispense a substance into the gastrointestinal tract (e.g., through theuse of optional opening 116 and optional rotating assembly 118 (FIG.2)), or may obtain a fluid sample from the environment external to thehousing of ingestible device 100 (e.g., through the use of optionalopening 116 and optional rotating assembly 118 (FIG. 2)). For instance,ingestible device 100 may be configured to obtain a sample in responseto determining that a number of detected muscle contractions isabnormal, and differs greatly from the expected number. As anotherexample, ingestible device 100 may be configured to deliver a substanceinto the GI tract (such as a medicament), in response to determiningthat the detected muscle contractions are consistent with a functioningGI tract in a healthy individual.

It will be understood that the steps and descriptions of the flowchartsof this disclosure, including FIG. 9, are merely illustrative. Any ofthe steps and descriptions of the flowcharts, including FIG. 9, may bemodified, omitted, rearranged, performed in alternate orders or inparallel, two or more of the steps may be combined, or any additionalsteps may be added, without departing from the scope of the presentdisclosure. For example, the ingestible device 100 may calculate themean and the standard deviation of multiple data sets in parallel (e.g.,multiple data sets, each one corresponding to a different wavelength ofreflectance or different sensing sub-unit used to detect thereflectance) in order to speed up the overall computation time.Furthermore, it should be noted that the steps and descriptions of FIG.9 may be combined with any other system, device, or method described inthis application, and any of the ingestible devices or systems discussedin this application could be used to perform one or more of the steps inFIG. 9.

FIG. 10 is a plot illustrating data collected during an exampleoperation of an ingestible device, which may be used when detecting atransition from a duodenum to a jejunum, in accordance with someembodiments of the disclosure. Diagram 1000 depicts a time domain plot1002 of a data set of reflectance levels measured by an ingestibledevice (e.g., the second subset of data discussed in relation to 908 ofFIG. 9). In some embodiments, ingestible device 100 may be configured togather data points at semi-regular intervals approximately 0.5 secondsapart. By comparison, diagram 1050 depicts a frequency domain plot 1004of the same data set of reflectance levels measured by an ingestibledevice (e.g., as a result of ingestible device 100 calculating afrequency spectrum at 910 of FIG. 9). In some embodiments, ingestibledevice 100 may be configured to calculate the frequency spectrum throughany convenient means.

In diagram 1050, the range of frequencies 1006 between 0.1 Hz and 0.2 Hzmay be the range of frequencies that ingestible device 100 searches inorder to detect muscle contractions. As shown in diagram 1050, there isa strong peak in the frequency domain plot 1004 around 0.14 Hz, which isconsistent with the frequency of peristaltic motion in a healthy humanindividual. In this case, an ingestible device 100 analyzing frequencydomain plot 1004 may be configured to determine that the data isconsistent with a detected muscle contraction (e.g., using a processsimilar to 912 of process 900 (FIG. 9)), and may store data (e.g., inmemory circuitry of PCB 120 (FIG. 2)) indicating that a musclecontraction has been detected. Because the muscle contraction wasdetected from the one-minute window of data ending at 118 minutes,ingestible device 100 may also store data indicating that the musclecontraction was detected at the 118-minute mark (i.e., which mayindicate that the ingestible device 100 was turned on and ingested bythe subject 118 minutes ago).

FIG. 11 is a plot illustrating muscle contractions detected by aningestible device over time, which may be used when determining alocation of an ingestible device as it transits through agastrointestinal (GI) tract, in accordance with some embodiments of thedisclosure. In some embodiments, ingestible device 100 may be configuredto detect muscle contractions, and store data indicative of when eachmuscle contraction is detected (e.g., as part of 914 of process 900(FIG. 9)). Plot 1100 depicts the detected muscle contractions 1106 overtime, with each muscle contraction being represented by a vertical linereaching from “0” to “1” on the y-axis.

At 1102, around the 10-minute mark, ingestible device 100 first entersthe duodenum (e.g., as determined by ingestible device 100 performingprocess 600 (FIG. 6)). Shortly thereafter, at 1108, ingestible device100 begins to detect several muscle contractions 1106 in quicksuccession, which may be indicative of the strong peristaltic waves thatform in the jejunum (e.g., jejunum 314 (FIG. 3)). Later, around 1110,ingestible device 100 continues to detect intermittent musclecontractions, which may be consistent with an ingestible device 100within the ileum. Finally, at 1104, ingestible device 100 transitionsout of the small intestine, and into the cecum. Notably, ingestibledevice 100 detects more frequent muscle contractions in the jejunumportion of the small intestine as compared to the ileum portion of thesmall intestine, and ingestible device 100 does not measure any musclecontractions after having exited the small intestine. In someembodiments, ingestible device 100 may incorporate this information intoa localization process. For example, ingestible device 100 may beconfigured to detect a transition from a jejunum to an ileum in responseto determining that a frequency of detected muscle contractions (e.g.,the number of muscle contractions measured in a given 10-minute window)has fallen below a threshold number. As another example, ingestibledevice 100 may be configured to detect a transition from an ileum to acecum in response to determining that no muscle contractions have beendetected for a threshold period of time. It is understood that theseexamples are intended to be illustrative, and not limiting, and thatmeasurements of muscle contractions may be combined with any of theother processes, systems, or methods discussed in this disclosure.

FIG. 12 is a flowchart 1200 for certain embodiments for determining atransition of the device from the jejunum to the ileum. It is to benoted that, in general, the jejunum is redder and more vascular than theileum. Moreover, generally, in comparison to the ileum, the jejunum hasa thicker intestine wall with more messentary fat. These differencesbetween the jejunum and the ileum are expected to result in differencesin optical responses in the jejunum relative to the ileum. Optionally,one or more optical signals may be used to investigate the differencesin optical responses. For example, the process can include monitoring achange in optical response in reflected red light, blue light, greenlight, ratio of red light to green light, ratio of red light to bluelight, and/or ratio of green light to blue light. In some embodiments,reflected red light is detected in the process.

Flowchart 1200 represents a single sliding window process. In step 1210,the jejenum reference signal is determined based on optical reflection.Typically, this signal is as the average signal (e.g., reflected redlight) over a period of time since the device was determined to enterthe jejenum. The period of time can be, for example, from five minutesto 40 minutes (e.g., from 10 minutes to 30 minutes, from 15 minutes to25 minutes). In step 1220, the detected signal (e.g., reflected redlight) just after the period of time used in step 1210 is normalized tothe reference signal determined in step 1210. In step 1230, the signal(e.g., reflected red light) is detected. In step 1240, the mean signaldetected based on the single sliding window is compared to a signalthreshold. The signal threshold in step 1240 is generally a fraction ofthe reference signal of the jejenum reference signal determined in step1210. For example, the signal threshold can be from 60% to 90% (e.g.,from 70% to 80%) of the jejenum reference signal. If the mean signalexceeds the signal threshold, then the process determines that thedevice has entered the ileum at step 1250. If the mean signal does notexceed the signal threshold, then the process returns to step 1230.

FIG. 13 is a flowchart 1200 for certain embodiments for determining atransition of the device from the jejunum to the ileum using a twosliding window process. In step 1310, the jejenum reference signal isdetermined based on optical reflection. Typically, this signal is as theaverage signal (e.g., reflected red light) over a period of time sincethe device was determined to enter the jejenum. The period of time canbe, for example, from five minutes to 40 minutes (e.g., from 10 minutesto 30 minutes, from 15 minutes to 25 minutes). In step 1320, thedetected signal (e.g., reflected red light) just after the period oftime used in step 1310 is normalized to the reference signal determinedin step 1310. In step 1330, the signal (e.g., reflected red light) isdetected. In step 1340, the mean difference in the signal detected basedon the two sliding windows is compared to a signal threshold. The signalthreshold in step 1340 is based on whether the mean difference in thedetected signal exceeds a multiple (e.g., from 1.5 times to five times,from two times to four times) of the detected signal of the firstwindow. If signal threshold is exceeded, then the process determinesthat the device has entered the ileum at step 1350. If the signalthreshold is not exceeded, then the process returns to step 1330.

FIG. 14 is a flowchart 1400 for a process for certain embodiments fordetermining a transition of the device from the ileum to the cecum. Ingeneral, the process involves detecting changes in the reflected opticalsignal (e.g., red light, blue light, green light, ratio of red light togreen light, ratio of red light to blue light, and/or ratio of greenlight to blue light). In some embodiments, the process includesdetecting changes in the ratio of reflected red light to reflected greenlight, and also detecting changes in the ratio of reflected green lightto reflected blue light. Generally, in the process 1400, the slidingwindow analysis (first and second windows) discussed with respect toprocess 600 is continued.

Step 1410 includes setting a first threshold in a detected signal, e.g.,ratio of detected red light to detected green light, and setting asecond threshold for the coefficient of variation for a detected signal,e.g., the coefficient of variation for the ratio of detected green lightto detected blue light. The first threshold can be set to a fraction(e.g., from 0.5 to 0.9, from 0.6 to 0.8) of the average signal (e.g.,ratio of detected red light to detected green light) in the firstwindow, or a fraction (e.g., from 0.4 to 0.8, from 0.5 to 0.7) of themean difference between the detected signal (e.g., ratio of detected redlight to detected green light) in the two windows. The second thresholdcan be set to 0.1 (e.g., 0.05, 0.02).

Step 1420 includes detecting the signals in the first and second windowsthat are to be used for comparing to the first and second thresholds.

Step 1430 includes comparing the detected signals to the first andsecond thresholds. If the corresponding value is not below the firstthreshold or the corresponding value is not below the second threshold,then it is determined that the device has not left the ileum and enteredthe cecum, and the process returns to step 1420. If the correspondingvalue is below the first threshold and the corresponding value is belowthe second threshold, then it is determined that the device has left theileum and entered the cecum, and the proceeds to step 1440.

Step 1450 includes determining whether it is the first time that thatthe device was determined to leave the ileum and enter the cecum. If itis the first time that the device was determined to leave the ileum andenter the cecum, then the process proceeds to step 1460. If it is notthe first time that the device has left the ileum and entered the cecum,then the process proceeds to step 1470.

Step 1460 includes setting a reference signal. In this step the opticalsignal (e.g., ratio of detected red light to detected green light) as areference signal.

Step 1470 includes determining whether the device may have left thececum and returned to the ileum. The device is determined to have leftthe cecum and returned to the ileum if the corresponding detected signal(e.g., ratio of detected red light to detected green light) isstatistically comparable to the reference signal (determined in step1460) and the coefficient of variation for the corresponding detectedsignal (e.g., ratio of detected green light to detected blue light)exceeds the second threshold. If it is determined that the device mayhave left the cecum and returned to the ileum, the process proceeds tostep 1480.

Step 1480 includes continuing to detect the relevant optical signals fora period of time (e.g., at least one minute, from five minutes to 15minutes).

Step 1490 includes determining whether the signals determined in step1480 indicate (using the methodology discussed in step 1470) that thedevice re-entered the ileum. If the signals indicate that the devicere-entered the ileum, the process proceeds to step 1420. If the signalsindicate that the device is in the cecum, the process proceeds to step1492.

Step 1492 includes continuing to monitor the relevant optical signalsfor a period of time (e.g., at least 30 minutes, at least one hour, atleast two hours).

Step 1494 includes determining whether the signals determined in step1492 indicate (using the methodology discussed in step 1470) that thedevice re-entered the ileum. If the signals indicate that the devicere-entered the ileum, the process proceeds to step 1420. If the signalsindicate that the device is in the cecum, the process proceeds to step1496.

At step 1496, the process determines that the device is in the cecum.

FIG. 15 is a flowchart 1500 for a process for certain embodiments fordetermining a transition of the device from the cecum to the colon. Ingeneral, the process involves detecting changes in the reflected opticalsignal (e.g., red light, blue light, green light, ratio of red light togreen light, ratio of red light to blue light, and/or ratio of greenlight to blue light). In some embodiments, the process includesdetecting changes in the ratio of reflected red light to reflected greenlight, and also detecting changes in the ratio of reflected blue light.Generally, in the process 1500, the sliding window analysis (first andsecond windows) discussed with respect to process 1400 is continued.

In step 1510, optical signals (e.g., the ratio of reflected red signalto reflected green signal, and reflected blue signal) are collected fora period of time (e.g., at least one minute, at least five minutes, atleast 10 minutes) while the device is in the cecum (e.g., during step1480). The average values for the recorded optical signals (e.g., theratio of reflected red signal to reflected green signal, and reflectedblue signal) establish the cecum reference signals.

In step 1520, the optical signals are detected after it has beendetermined that the device entered the cecum (e.g., at step 1440). Theoptical signals are normalized to the cecum reference signals.

Step 1530 involves determining whether the device has entered the colon.This includes determining whether any of three different criteria aresatisfied. The first criterion is satisfied if the mean difference inthe ratio of a detected optical signal (e.g., ratio of detected redsignal to the detected green) is a multiple greater than one (e.g., 2×,3×, 4×) the standard deviation of the corresponding signal (e.g., ratioof detected red signal to the detected green) in the second window. Thesecond criterion is satisfied if the mean of a detected optical signal(e.g., a ratio of detected red light to detected green light) exceeds agiven value (e.g., exceeds one). The third criterion is satisfied if thecoefficient of variation of an optical signal (e.g., detected bluelight) in the first window exceeds a given value (e.g., exceeds 0.2). Ifany of the three criteria are satisfied, then the process proceeds tostep 1540. Otherwise, none of the three criteria are satisfied, theprocess returns to step 1520.

For illustrative purposes the disclosure focuses primarily on a numberof different example embodiments of an ingestible device, and exampleembodiments of methods for determining a location of an ingestibledevice within a GI tract. However, the possible ingestible devices thatmay be constructed are not limited to these embodiments, and variationsin the shape and design may be made without significantly changing thefunctions and operations of the device. Similarly, the possibleprocedures for determining a location of the ingestible device withinthe GI tract are not limited to the specific procedures and embodimentsdiscussed (e.g., process 500 (FIG. 5), process 600 (FIG. 6), process 900(FIG. 9), process 1200 (FIG. 12), process 1300 (FIG. 13), process 1400(FIG. 14) and process 1500 (FIG. 15)). Also, the applications of theingestible devices described herein are not limited merely to gatheringdata, sampling and testing portions of the gastrointestinal tract, ordelivering medicament. For example, in some embodiments the ingestibledevice may be adapted to include a number of chemical, electrical, oroptical diagnostics for diagnosing a number of diseases. Similarly, anumber of different sensors for measuring bodily phenomenon or otherphysiological qualities may be included on the ingestible device. Forexample, the ingestible device may be adapted to measure elevated levelsof certain chemical compounds or impurities in the gastrointestinaltract, or the combination of localization, sampling, and appropriatediagnostic and assay techniques incorporated into a sampling chamber maybe particularly well suited to determine the presence of smallintestinal bacterial overgrowth (SIBO).

At least some of the elements of the various embodiments of theingestible device described herein that are implemented via software(e.g., software executed by control circuitry within PCB 120 (FIG. 2))may be written in a high-level procedural language such as objectoriented programming, a scripting language or both. Accordingly, theprogram code may be written in C, C++ or any other suitable programminglanguage and may comprise modules or classes, as is known to thoseskilled in object oriented programming. Alternatively, or in addition,at least some of the elements of the embodiments of the ingestibledevice described herein that are implemented via software may be writtenin assembly language, machine language or firmware as needed. In eithercase, the language may be a compiled or an interpreted language.

At least some of the program code used to implement the ingestibledevice can be stored on a storage media or on a computer readable mediumthat is readable by a general or special purpose programmable computingdevice having a processor, an operating system and the associatedhardware and software that is necessary to implement the functionalityof at least one of the embodiments described herein. The program code,when read by the computing device, configures the computing device tooperate in a new, specific and predefined manner in order to perform atleast one of the methods described herein.

Furthermore, at least some of the programs associated with the systems,devices, and methods of the example embodiments described herein arecapable of being distributed in a computer program product comprising acomputer readable medium that bears computer usable instructions for oneor more processors. The medium may be provided in various forms,including non-transitory forms such as, but not limited to, one or morediskettes, compact disks, tapes, chips, and magnetic and electronicstorage. In some embodiments, the medium may be transitory in naturesuch as, but not limited to, wire-line transmissions, satellitetransmissions, internet transmissions (e.g. downloads), media, digitaland analog signals, and the like. The computer useable instructions mayalso be in various formats, including compiled and non-compiled code.

The techniques described above can be implemented using software forexecution on a computer. For instance, the software forms procedures inone or more computer programs that execute on one or more programmed orprogrammable computer systems (which may be of various architecturessuch as distributed, client/server, or grid) each including at least oneprocessor, at least one data storage system (including volatile andnon-volatile memory and/or storage elements), at least one input deviceor port, and at least one output device or port.

The software may be provided on a storage medium, such as a CD-ROM,readable by a general or special purpose programmable computer ordelivered (encoded in a propagated signal) over a communication mediumof a network to the computer where it is executed. All of the functionsmay be performed on a special purpose computer, or using special-purposehardware, such as coprocessors. The software may be implemented in adistributed manner in which different parts of the computation specifiedby the software are performed by different computers. Each such computerprogram is preferably stored on or downloaded to a storage media ordevice (e.g., solid state memory or media, or magnetic or optical media)readable by a general or special purpose programmable computer, forconfiguring and operating the computer when the storage media or deviceis read by the computer system to perform the procedures describedherein. The inventive system may also be considered to be implemented asa computer-readable storage medium, configured with a computer program,where the storage medium so configured causes a computer system tooperate in a specific and predefined manner to perform the functionsdescribed herein.

Methods and Mechanisms of Delivery

FIG. 16 provides an example mock-up diagram illustrating aspects of astructure of an ingestible device 1600 for delivering a dispensablesubstance, such as a formulation of a therapeutic agent describedherein, according to some embodiments described herein. In someembodiments, the ingestible device 1600 may generally be in the shape ofa capsule, a pill or any swallowable form that may be orally consumed byan individual. In this way, the ingestible device 1600 may be ingestedby a patient and may be prescribed by healthcare practitioners andpatients.

The ingestible device 1600 includes a housing 1601 that may take a shapesimilar to a capsule, a pill, and/or the like, which may include twoends 1602 a-b. The housing 1601 may be designed to withstand thechemical and mechanical environment of the GI tract (e.g., effects ofmuscle contractile forces and concentrated hydrochloric acid in thestomach). A broad range of materials that may be used for the housing1601. Examples of these materials include, but are not limited to,thermoplastics, fluoropolymers, elastomers, stainless steel and glasscomplying with ISO 10993 and USP Class VI specifications forbiocompatibility; and any other suitable materials and combinationsthereof.

In some embodiment, the wall of the housing 1601 may have a thickness of0.5 mm-1 mm, which is sufficient to sustain an internal explosion (e.g.,caused by hydrogen ignition or over pressure inside the housing).

The housing 1601 may or may not have a pH-sensitive enteric coating todetect or otherwise be sensitive to a pH level of the environmentexternal to the ingestible device. As discussed elsewhere in theapplication in more detail, the ingestible device 1600 may additionallyor alternatively include one more sensors, e.g., temperature sensor,optical sense.

The housing 1601 may be formed by coupling two enclosure portionstogether. The ingestible device 1600 may include an electronic componentwithin the housing 1600. The electronic component may be placedproximally to an end 1602 b of the housing, and includes a printedcircuit board (PCB), a battery, an optical sensing unit, and/or thelike.

The ingestible device 1600 further includes a gas generating cell 1603that is configured to generate gas and thus cause an internal pressurewithin the housing 1601. In some embodiments, the gas generating cellmay include or be connected to a separate channel or valve of theingestible device such that gas may be release through the channel orvalve to create a motion to alter the position of the ingestible devicewithin the GI tract. Such gas release can also be used to position theingestible device relative to the intestinal lining. In anotherembodiment, gas may be released through the separate channel or valve toalter the surface orientation of the intestinal tissue prior to deliveryof the dispensable substance.

A traveling plunger 1604 may be placed on top of the gas generating cell1603 within the housing 1601. The traveling plunger 1604 is a membranethat separates the gas generating cell 1603 and a storage reservoir thatstores the dispensable substance 1605. In some embodiments, thetraveling plunger 1604 may be a movable piston. In some embodiments, thetraveling plunger 1604 may instead be a flexible membrane such as butnot limited to a diaphragm. In some embodiments, the traveling plunger1604, which may have the form of a flexible diaphragm, may be placedalong an axial direction of the housing 1601, instead of being placed ontop of the gas generating cell 1603. The traveling plunger or themembrane 1604 may move (when the membrane 1604 is a piston) or deform(when the membrane 1604 is a diaphragm) towards a direction of the end1602 a of the housing, when the gas generating cell 1603 generates gasto create an internal pressure that pushes the membrane 1604. In thisway, the membrane or traveling plunger 1604 may push the dispensablesubstance 1605 out of the housing via a dispensing outlet 1607.

The housing 1601 may include a storage reservoir storing one or moredispensable substances 1605 adjacent to the traveling plunger 1604. Thedispensable substance 1605 may be a therapeutic or medical agent thatmay take a form of a powder, a compressed powder, a fluid, a semi-liquidgel, or any other dispensable or deliverable form. The delivery of thedispensable substance 1605 may take a form such as but not limited tobolus, semi-bolus, continuous, systemic, burst drug delivery, and/or thelike. In some embodiments, a single bolus is delivered proximate to thedisease location. In some embodiments, more than one bolus is releasedat one location or more than one location. In some embodiments therelease of more than one bolus is triggered according to apre-programmed algorithm. In some embodiments the release profile iscontinuous. In some embodiments the release profile is time-based. Insome embodiments the release profile is location-based. In someembodiments, the amount delivered is based on the severity and/or extentof the disease in the following manner. In some embodiments, the bolusis delivered in one or more of the following locations: stomach;duodenum; proximal jejunum; ileum; cecum; ascending colon; transversecolon; descending colon. In some embodiments the dispensable substanceis a small molecule therapeutic that is released in the cecum and/orother parts of the large intestine. Small molecules that areadministered by typical oral routes are primarily absorbed in the smallintestine, with much lower absorption taking place in the largeintestine (outside of the rectum). Accordingly, an ingestible devicethat is capable of releasing a small molecule selectively in the largeintestine (e.g., the cecum) with resulting low systemic levels (evenwhen high doses are used) is attractive for subjects with inflammatorybowel disease in the large intestine.

In some embodiments, the storage reservoir may include multiplechambers, and each chamber stores a different dispensable substance. Forexample, the different dispensable substances can be released at thesame time via the dispensing outlet 1607. Alternatively, the multiplechambers may take a form of different layers within the storagereservoir such that the different dispensable substance from eachchamber is delivered sequentially in an order. In one example, each ofthe multiple chambers is controlled by a separate traveling plunger,which may be propelled by gas generation. The electronic component maycontrol the gas generating cell 1603 to generate gas to propel aspecific traveling plunger, e.g., via a separate gas generation chamber,etc., to deliver the respective substance. In some embodiments, thecontent of the multiple chambers may be mixed or combined prior torelease, for example, to activate the drug.

The ingestible device 1600 may include a dispensing outlet 1607 at oneend 1602 a of the housing 1601 to direct the dispensable substance 105out of the housing. The dispensing outlet 1607 may include an exitvalve, a slit or a hole, a jet injection nozzle with a syringe, and/orthe like. When the traveling plunger 1604 moves towards the end 1602 aof the housing 1601, an internal pressure within the storage reservoirmay increase and push the dispensing outlet to be open to let thedispensable substance 1605 be released out of the housing 1601.

In an embodiment, a pressure relief device 1606 may be placed within thehousing 1601, e.g., at the end 1602 a of the housing 1601.

In some embodiments, the housing 1601 may include small holes (e.g.,with a diameter smaller than 2 mm), e.g., on the side of the housing1601, or at the end 1602 a to facilitate loading the dispensablesubstance into the storage reservoir.

In some embodiments, a feedback control circuit (e.g., a feedbackresistor, etc.) may be added to send feedback from the gas generatingcell 1603 to the electronic component such that when the internalpressure reaches a threshold level, the electronic component may controlthe gas generating cell 1603 to turn off gas generation, or to activateother safety mechanism (e.g., feedback-controlled release valve, etc.).For example, an internal pressure sensor may be used to measure theinternal pressure within the ingestible device and generate feedback tothe feedback control circuit.

FIG. 17 provides an example diagram illustrating aspects of a mechanismfor a gas generating cell 1603 configured to generate a gas to dispensea substance, according to some embodiments described herein. As shown inFIG. 17, the gas generating cell 1603 generates a gas 1611 which canpropel the dispensable substance 1605 out of the dispensing outlet 1607.A variable resistor 1608 may be connected to a circuit with the gasgenerating cell 1603 such that the variable resistor 1608 may be used tocontrol an intensity and/or an amount of gas 1611 (e.g., hydrogen)generated by the cell 1603. Specifically, the gas generating cell 1603may be a battery form factor cell that is capable of generating hydrogenwhen a resistor is applied. In this way, as the gas generating cell 1603only needs the use of a resistor only without any active powerrequirements, the gas generating cell 1603 may be integrated into aningestible device such as a capsule with limited energy/power available.For example, the gas generating cell 1603 may be compatible with acapsule at a size of 26 mm×13 mm or smaller.

In some embodiments, based on the elution rate of gas from the cell, andan internal volume of the ingestible device, it may take time togenerate sufficient gas 1611 to deliver the substance 1605, and the timerequired may be 30 seconds or longer. For example, the time to generatea volume of hydrogen equivalent to 5004 of fluid would be approximately5 minutes. A longer period of time may be needed based upon non-idealconditions within the ingestible device, such as friction, etc. Thus,given that the production of gas (e.g., hydrogen) may take time, gasgeneration may need to start prior to the ingestible device arriving atthe site of delivery to build pressure up within the device. Theingestible device may then need to know when it is approaching the siteof delivery. For example, the device may start producing gas on an“entry transition,” which is determined by temperature, so as to produceenough gas to be close to the pressure high enough to deliver thedispensable substance. The ingestible device may then only startproducing gas again when it arrives at the site of delivery, which willcause the internal pressure within the ingestible device to reach alevel required by the dispensing outlet to release the dispensablesubstance. Also, for regio-specific delivery, the ingestible device mayestimate the time it takes to build up enough pressure to deliver thedispensable substance before the ingestible device arrives at a specificlocation, to activate gas generation.

For example, for systemic delivery, when an internal volume of theingestible device is around 500 μL, a gas generation time of 2 hours, aninitial pressure of approximately 300 pound per square inch absolute(psia) may be generated, with higher and lower pressures possible. Thegenerated pressure may drop when air enters the storage reservoir whichwas previously occupied by the dispensable substance during thedispensing process. For systemic drug delivery, a force with a generatedpressure of approximately 100 to 360 pound per square inch (psi) may berequired for dermal penetration, e.g., to penetrate the mucosa orepithelial layer. The pressure may also vary depending on the nozzledesign at the dispensing outlet, fluid viscosity, and surrounding tissueproximity and properties.

The gas 1611 that may be generated for a continuous delivery of drug(e.g., 1 cc H₂ in 4 hours, 16 breaths per minute at 0.5 L tidal volume)may equate to 1 cc hydrogen in approximately 2000 L of exhaled air, orapproximately 0.5 ppm H2, which is below physiologic values of exhaledhydrogen. Reducing this time to 10 minutes equates to approximately 13ppm hydrogen. Thus, due to the length of intestine that may be coveredduring this time period, the ingestible device may possess a higherlocalized value than physiologic.

FIGS. 18 and 19, disclosed in U.S. Provisional Application No.62/385,553, incorporated by reference herein in its entirety,illustrates an example of an ingestible device for localized delivery ofpharmaceutical compositions disclosed herein, in accordance withparticular implementations. The ingestible device 1600 includes a pistonor drive element 1634 to push for drug delivery, in accordance withparticular implementations described herein. The ingestible device 1600may have one or more batteries 1631 placed at one end 1602 a of ahousing 1601 to provide power for the ingestible device 1600. A printedcircuit board (PCB) 1632 may be placed adjacent to a battery or otherpower source 1631, and a gas generating cell 1603 may be mounted on orabove the PCB 1632. The gas generating cell 1603 may be sealed from thebottom chamber (e.g., space including 1631 and 1632) of the ingestibledevice 1600. A movable piston 1634 may be placed adjacent to the gasgenerating cell 1603. In this way, gas generation from the gasgenerating cell 1603 may propel a piston 1634 to move towards anotherend 1602 b of the housing 1601 such that the dispensable substance in areservoir compartment 1635 can be pushed out of the housing through adispensing outlet 1607, e.g., the movement is shown at 1636, with thepiston 1634 at a position after dispensing the substance. The dispensingoutlet 1607 may comprise a plug. The reservoir compartment 1635 canstore the dispensable substance (e.g., drug substance), or alternativelythe reservoir compartment can house a storage reservoir 1661 whichcomprises the dispensable substance. The reservoir compartment 1635 orstorage reservoir 1661 may have a volume of approximately 600 μL or evenmore dispensable substance, which may be dispensed in a single bolus, orgradually over a period of time.

The battery cells 1631 may have a height of 1.65 mm each, and one tothree batteries may be used. The height of the piston may be reducedwith custom molded part for around 1.5 mm to save space. If the gasgenerating cell 1603 is integrated with the piston 1634, the overallheight of the PCB, batteries and gas generating cell in total can bereduced to around 5 mm, thus providing more space for drug storage. Forexample, for an ingestible device of 7.8 mm in length (e.g., from end1602 a to the other end 1602 b), a reservoir compartment 1635 or astorage reservoir 1661 of approximately 600 μL may be used for drugdelivery. For another example, for an ingestible device of 17.5 mm inlength, a reservoir compartment 1635 or a storage reservoir 1661 ofapproximately 1300 μL may be used for drug release. In someimplementations, at the reservoir 1635 or 1661 for storing atherapeutically effective amount of the TLR agonist forms at least aportion of the device housing 1601. The therapeutically effective amountof the TLR agonist can be stored in the reservoir 1635 or 1661 at aparticular pressure, for example, determined to be higher than apressure inside the GI tract so that once the reservoir 1635 or 1661 isin fluid communication with the GI tract, the TLR agonist isautomatically released. In certain implementations, the reservoircompartment 1635 includes a plurality of chambers, and each of theplurality of the chambers stores a different dispensable substance or adifferent storage reservoir 1661.

In certain embodiments, the storage reservoir 1661 is a compressiblecomponent or has compressible side walls. In particular embodiments, thecompressible component can be composed, at least in part, or coated(e.g., internally) with polyvinyl chloride (PVC), silicone, DEHP(di-2-ethylhexyl phthalate), Tyvek, polyester film, polyolefin,polyethylene, polyurethane, or other materials that inhibit the TLRagonist from sticking to the reservoir and provide a sterile reservoirenvironment for the TLR agonist. The storage reservoir 1661 can behermetically sealed. The reservoir compartment 1635 or storage reservoir1661 can be configured to store TLR agonist in quantities in the rangeof 0.01 mL-2 mL, such as 0.05 mL-2 mL, such as 0.05 mL-2 mL, such as 0.6mL-2 mL. In some embodiments, the storage reservoir 1661 is attachableto the device housing 1601, for example, in the reservoir compartment.Accordingly, the storage reservoir 1635 can be loaded with the TLRagonist prior to being positioned in and/or coupled to the ingestibledevice housing 1601. The ingestible device housing 1601 includes one ormore openings configured as a loading port to load the dispensablesubstance into the reservoir compartment. In another embodiment, theingestible device housing 1601 includes one or more openings configuredas a vent.

As noted above, in some embodiments, a storage reservoir (optionally,containing a TLR agonist, such as a therapeutically effective amount ofTLR agonist) is attachable to an ingestible device. In general, in suchembodiments the storage reservoir and ingestible device can be designedin any appropriate fashion so that the storage reservoir can attach tothe ingestible device when desired. Examples of designs include astorage reservoir that fits entirely within the ingestible device (e.g.,in the ingestible device so that the storage reservoir is sealed withinthe device at the time the device is ingested by a subject), a storagereservoir that fits partially within the ingestible device, and astorage reservoir that is carried by the housing of the device. In someembodiments, the storage reservoir snap fits with the ingestible device.In certain embodiments, the storage reservoir is friction fit with theingestible device. In some embodiments, the storage reservoir is heldtogether with the ingestible device via a biasing mechanism, such as oneor more springs, one or more latches, one or more hooks, one or moremagnets, and/or electromagnetic radiation. In certain embodiments, thestorage reservoir can be a piercable member. In some embodiments, theingestible device has a sleeve into which the storage reservoir securelyfits. In some embodiments, the storage reservoir is disposed in/on aslidable track/groove so that it can move onto a piercing needle whendelivery of the therapeutic agent is desired. In certain embodiments,the storage reservoir is made of a soft plastic coating, which iscontacted with a needle at any orientation to deliver the therapeuticagent when desired. Generally, the storage reservoir can be made of oneor more appropriate materials, such as, for example, one or moreplastics and/or one or more metals or alloys. Exemplary materialsinclude silicone, polyvinyl chloride, polycarbonate and stainless steel.Optionally, the design may be such that the storage reservoir carriessome or all of the electrical componentry to be used by the ingestibledevice. Although the foregoing discussion relates to one storagereservoir, it is to be understood that an ingestible device can bedesigned to carry any desired number (e.g., two, three, four, five)storage reservoirs. Different storage reservoirs can have the same ordifferent designs. In some embodiments, the ingestible device (whenfully assembled and packaged) satisfies the regulatory requirements formarketing a medical device in one or more jurisdictions selected fromthe United States of America, the European Union or any member statethereof, Japan, China, Brazil, Canada, Mexico, Colombia, Argentina,Chile, Peru, Russia, the UK, Switzerland, Norway, Turkey, Israel, anymember state of the Gulf Cooperative Council, South Africa, India,Australia, New Zealand, South Korea, Singapore, Thailand, thePhilippines, Malaysia, Viet Nam, and Indonesia, Taiwan and Hong Kong.

In certain embodiments, the ingestible device housing 1601 includes oneor more actuation systems (e.g., gas generating cell 1603) for pumpingthe TLR agonist from the reservoir 1635. In some embodiments, theactuation system can include a mechanical, electrical,electromechanical, hydraulic, and/or fluid actuation system. Forexample, a chemical actuation means may use chemical reaction of mixingone or more reagents to generate a sufficient volume of gas to propelthe piston or drive element 1634 for drug release. The actuation systemcan be integrated into the reservoir compartment 1635 or can be anauxiliary system acting on or outside of the reservoir compartment 1635.For example, the actuation system can include pumping system forpushing/pulling the TLR agonist out of the reservoir compartment 1635 orthe actuation system can be configured to cause the reservoircompartment 1635 to change structurally so that the volume inside of thereservoir compartment 1635 changes, thereby dispensing the TLR agonistfrom the reservoir compartment 1635. The actuation system can include anenergy storage component such as a battery or a capacitor for poweringthe actuation system. The actuation system can be actuated via gaspressure or a system storing potential energy, such as energy from anelastic reservoir component being expanded during loading of thereservoir and after being positioned in the ingestible device housing1601 being subsequently released from the expanded state when theingestible device housing is at the location for release within the GItract. In certain embodiments, the reservoir compartment 1635 caninclude a membrane portion, whereby the TLR agonist is dispensed fromthe reservoir compartment 1635 or storage reservoir 1661 via osmoticpressure.

In particular embodiments the storage reservoir 1661 is in a form of abellow that is configured to be compressed via a pressure from the gasgenerating cell. The TLR agonist may be loaded into the bellow, whichmay be compressed by gas generation from the gas generating cell orother actuation means to dispense the dispensable substance through thedispensing outlet 1607 and out of the housing 1601. In some embodiments,the ingestible device includes a capillary plate placed between the gasgenerating cell and the first end of the housing, and a wax seal betweenthe gas generating cell and the reservoir, wherein the wax seal isconfigured to melt and the dispensable substance is pushed through thecapillary plate by a pressure from the gas generating cell. The shape ofthe bellow may aid in controlled delivery. The reservoir compartment1635 includes a dispensing outlet, such as a valve or dome slit 1662extending out of an end of the housing 1601, in accordance withparticular implementations. Thus when the bellow is being compressed,the dispensable substance may be propelled out of the bellow through thevalve or the dome slit.

In certain embodiments, the reservoir compartment 1635 includes one ormore valves (e.g. a valve in the dispensing outlet 1607) that areconfigured to move or open to fluidly couple the reservoir compartment1635 to the GI tract. In certain embodiments, a housing wall of thehousing 1601 can form a portion of the reservoir compartment 1635. Incertain embodiments, the housing walls of the reservoir serve as agasket. One or more of the one or more valves are positioned in thehousing wall of the device housing 1601, in accordance with particularimplementations. One or more conduits may extend from the reservoir 1635to the one or more valves, in certain implementations.

In certain embodiments, a housing wall of the housing 1601 can be formedof a material that is configured to dissolve, for example, in responseto contact at the disease site. In certain embodiments, a housing wallof the housing 1601 can be configured to dissolve in response to achemical reaction or an electrical signal. The one or more valves and/orthe signals for causing the housing wall of the housing 1601 to dissolveor dissipate can be controlled by one or more processors or controllerspositioned on PCB 1632 in the device housing 1601. The controller iscommunicably coupled to one or more sensors or detectors configured todetermine when the device housing 1601 is proximate to a disease site.The sensors or detectors comprise a plurality of electrodes comprising acoating, in certain implementations. Releasing of the TLR agonist fromthe reservoir compartment 1635 is triggered by an electric signal fromthe electrodes resulting from the interaction of the coating with theone or more sites of disease site. The one or more sensors can include achemical sensor, an electrical sensor, an optical sensor, anelectromagnetic sensor, a light sensor, and/or a radiofrequency sensor.

In particular embodiments, the device housing 1601 can include one ormore pumps configured to pump the therapeutically effective amount ofthe TLR agonist from the reservoir compartment 1635. The pump iscommunicably coupled to the one or more controllers. The controller isconfigured to activate the pump in response to detection by the one ormore detectors of the disease site and activation of the valves to allowthe reservoir 1635 to be in fluid communication with the GI tract. Thepump can include a fluid actuated pump, an electrical pump, or amechanical pump.

In certain embodiments, the device housing 1601 comprises one or moreanchor systems for anchoring the device housing 1601 or a portionthereof at a particular location in the GI tract adjacent the diseasesite. In some embodiments, a storage reservoir comprises an anchorsystem, and the storage reservoir comprising a releasable substance isanchored to the GI tract. The anchor system can be activated by thecontroller in response to detection by the one or more detectors of thedisease site. In certain implementations, the anchor system includeslegs or spikes configured to extend from the housing wall(s) of thedevice housing 1601. The spikes can be configured to retract and/or canbe configured to dissolve over time. An example of an attachable devicethat becomes fixed to the interior surface of the GI tract is describedin PCT Patent Application PCT/US2015/012209, “Gastrointestinal SensorImplantation System”, filed Jan. 21, 2015, which is hereby incorporatedby reference herein in its entirety.

FIG. 20 provides an example structural diagram having a flexiblediaphragm 1665 that may deform towards the dispensing outlet 1607 whenthe gas generating cell 1603 generates gas. The dispensable substancemay then be propelled by the deformed diaphragm out of the housingthrough the dispensing outlet 1607. The dispensing outlet 1607 shown atFIG. 20 is in the form of a ring valve, however, any outlet design canbe applied.

In some embodiments, an ingestible device can have an umbrella-shapedexit valve structure as a dispensing outlet of the ingestible device.Optionally, an ingestible device can have a flexible diaphragm to deformfor drug delivery, and/or an integrated piston and gas generating cellsuch that the gas generating cell is movable with the piston to push fordrug delivery.

In certain embodiments, an ingestible device can be anchored within theintestine by extending hooks from the ingestible device after it hasentered the region of interest. For example, when the ingestible devicedetermines it has arrived at a location within the GI tract, the hookscan be actuated to extend outside of the ingestible device to catch inthe intestinal wall and hold the ingestible device in the respectivelocation. In some embodiments, the hook can pierce into the intestinalwall to hold the ingestible device 100 in place. The hooks can behollow. A hollow hook can be used to anchor the ingestible device and/orto dispense a substance from the dispensable substance, e.g., into theintestinal wall.

In some embodiments an ingestible device includes an intestinal gripperto grip a portion of the intestinal wall for delivering the dispensablesubstance. Such a gripper can include two or more arms configured to outof the device and close to grip a portion of the intestinal wall.

An injecting needle can be used with the anchoring arms to injectdispensable substance into the intestinal wall after a portion of theintestinal wall is gripped.

In some embodiments, when the gas generating cell generates gas topropel the piston to move towards the nozzle such that the dispensablesubstance can be pushed under the pressure to break a burst disc to beinjected via the nozzle.

In some embodiments, an ingestible device has a jet delivery mechanismwith enhanced usable volume of dispensable substance. For example, thenozzle may be placed at the center of the ingestible device, and gaschannels may be placed longitudinally along the wall of the ingestibledevice to transport gas from the gas generating cell to propel thepiston, which is placed at an end of the ingestible device.

In some embodiments, the ingestible device can use osmotic pressure toadhere a suction device of the ingestible device to the intestinal wall.For example, the ingestible device may have an osmotic mechanism thathas a chamber storing salt crystals. The chamber can include a meshplaced in proximate to a burst valve at one end of the chamber, and areverse osmosis (RO) membrane placed in proximate to a valve on theother end of the chamber. A suction device, e.g., two or more suctionfingers, is placed outside of the chamber with an open outlet exposed toluminal fluid in the GI tract. When the osmotic mechanism isinactivated, e.g., the valve is closed so that no luminal fluid is drawninto the osmotic chamber. When the osmotic mechanism is activated byopening the valve, luminal fluid enters the ingestible device through anoutlet of the suction device and enters the osmotic chamber through thevalve. The salt in the chamber is then dissolved into the fluid. The ROmembrane prevents any fluid to flow in the reverse direction, e.g., frominside the chamber to the valve. The fluid continues to flow until allthe salt contained in the chamber is dissolved or until intestinaltissue is drawn into the suction device. As luminal fluid keeps flowinginto the chamber, the solution of the luminal fluid with dissolved saltin the chamber may reduce osmotic pressure such that the suction forceat may also be reduced. In this way, suction of the intestinal tissuemay stall before the tissue is in contact with the valve to avoid damageto the intestinal tissue.

An ingestible device employing an osmotic mechanism can also include asuction device as illustrated. The suction device can be two or moresuction fingers 347 a-b disposed proximate to the outlet. The outlet canbe connected to a storage reservoir storing the dispensable substance(e.g., therapeutic agent). The storage reservoir can contact a piston(similar to 104 in FIG. 16), which can be propelled by pressuregenerated from the osmotic pump to move towards the outlet. The osmoticpump can be similar to the osmotic mechanism described in the precedingparagraph. A breakaway section can be placed in proximate to the otherend (opposite to the end where the outlet 107 is disposed) of theingestible device.

In some embodiments, tumbling suction by an ingestible device is used.Such an ingestible device does not require any electronics or otheractuation elements. Such an ingestible device may constantly,intermittently, or periodically tumble when travelling through theintestine. When the ingestible device tumbles to a position that theoutlet is in direct contact with the intestinal wall, a suction processsimilar to that described in the preceding paragraph may occur.Additional structural elements such as fins, flutes or the like may beadded to the outer wall of the ingestible device 100 to promote thetumbling motion.

In certain embodiments, the reservoir is an anchorable reservoir, whichis a reservoir comprising one or more anchor systems for anchoring thereservoir at a particular location in the GI tract adjacent the diseasesite. In certain embodiments, the anchor system includes legs or spikesor other securing means such as a piercing element, a gripping element,a magnetic-flux-guiding element, or an adhesive material, configured toextend from the anchorable reservoir of the device housing. The spikescan be configured to retract and/or can be configured to dissolve overtime. In some embodiments, the anchorable reservoir is suitable forlocalizing, positioning and/or anchoring. In some embodiments, theanchorable reservoir is suitable for localizing, and positioning and/oranchoring by an endoscope. In some embodiments, the anchorable reservoiris connected to the endoscope. In some embodiments, the anchorablereservoir is connected to the endoscope in a manner suitable for oraladministration. In some embodiments, the anchorable reservoir isconnected to the endoscope in a manner suitable for rectaladministration. Accordingly, provided herein in some embodiments is ananchorable reservoir is connected to an endoscope wherein the anchorablereservoir comprises a therapeutically effective amount of the TLRagonist. In some embodiments the endoscope is fitted with a spraycatheter.

Exemplary embodiments of anchorable reservoirs are as follows. In moreparticular examples of the following exemplary embodiments the reservoiris connected to an endoscope.

In one embodiment, the anchorable reservoir comprises an implant capsulefor insertion into a body canal to apply radiation treatment to aselected portion of the body canal. The reservoir includes a body memberdefining at least one therapeutic treatment material receiving chamberand at least one resilient arm member associated with the body memberfor removably engaging the body canal when the device is positionedtherein.

In one embodiment the anchorable reservoir has multiple suction portsand permits multiple folds of tissue to be captured in the suction portswith a single positioning of the device and attached together by atissue securement mechanism such as a suture, staple or other form oftissue bonding. The suction ports may be arranged in a variety ofconfigurations on the reservoir to best suit the desired resultingtissue orientation.

In some embodiments an anchorable reservoir comprises a tract stimulatorand/or monitor IMD comprising a housing enclosing electrical stimulationand/or monitoring circuitry and a power source and an elongated flexiblemember extending from the housing to an active fixation mechanismadapted to be fixed into the GI tract wall is disclosed. After fixationis effected, the elongated flexible member bends into a preformed shapethat presses the housing against the mucosa so that forces that wouldtend to dislodge the fixation mechanism are minimized. The IMD is fittedinto an esophageal catheter lumen with the fixation mechanism aimedtoward the catheter distal end opening whereby the bend in the flexiblemember is straightened. The catheter body is inserted through theesophagus into the GI tract cavity to direct the catheter distal end tothe site of implantation and fix the fixation mechanism to the GI tractwall. The IMD is ejected from the lumen, and the flexible member assumesits bent configuration and lodges the hermetically sealed housingagainst the mucosa. A first stimulation/sense electrode is preferably anexposed conductive portion of the housing that is aligned with the bendof the flexible member so that it is pressed against the mucosa. Asecond stimulation/sense electrode is located at the fixation site.

In some embodiments a reservoir for sensing one or more parameters of apatient is anchored to a tissue at a specific site and is released froma device, using a single actuator operated during a single motion. As anexample, a delivery device may anchor the capsule to the tissue site andrelease the reservoir from the delivery device during a single motion ofthe actuator.

In some embodiments a device is provided comprising: a reservoirconfigured to contain a fluid, the reservoir having at least one outletthrough which the fluid may exit the reservoir; a fluid contained withinthe reservoir; a primary material contained within the reservoir andhaving a controllable effective concentration in the fluid; and at leastone electromagnetically responsive control element located in thereservoir or in a wall of the reservoir and adapted for modifying thedistribution of the primary material between a first active form carriedin the fluid and a second form within the reservoir in response to anincident electromagnetic control signal, the effective concentrationbeing the concentration of the first active form in the fluid, wherebyfluid exiting the reservoir carries the primary material in the firstactive form at the effective concentration.

In some embodiments systems and methods are provided for implementing ordeploying medical or veterinary devices or reservoirs (a) operable foranchoring at least partly within a digestive tract, (b) small enough topass through the tract per vias naturales and including awireless-control component, (c) having one or more protrusionspositionable adjacent to a mucous membrane, (d) configured to facilitateredundant modes of anchoring, (e) facilitating a “primary” materialsupply deployable within a stomach for an extended and/or controllableperiod, (f) anchored by one or more adaptable extender modules supportedby a subject's head or neck, and/or (g) configured to facilitatesupporting at least a sensor within a subject's body lumen for up to aday or more.

In certain embodiments, the reservoir is attachable to an ingestibledevice. In certain embodiments, the ingestible device comprises ahousing and the reservoir is attachable to the housing. In certainembodiments, the attachable reservoir is also an anchorable reservoir,such as an anchorable reservoir comprising one or more anchor systemsfor anchoring the reservoir at a particular location in the GI tract asdisclosed hereinabove.

Accordingly, in certain embodiments, provided herein is a TLR agonistfor use in a method of treating a disease of the gastrointestinal tractas disclosed herein, wherein the TLR agonist is contained in a reservoirsuitable for attachment to a device housing, and wherein the methodcomprises attaching the reservoir to the device housing to form theingestible device, prior to orally administering the ingestible deviceto the subject.

In certain embodiments, provided herein is an attachable reservoircontaining a TLR agonist for use in a method of treating a disease ofthe gastrointestinal tract, wherein the method comprises attaching thereservoir to a device housing to form an ingestible device and orallyadministering the ingestible device to a subject, wherein the TLRagonist is released by device at a location in the gastrointestinaltract of the subject that is proximate to one or more sites of disease.

In certain embodiments, provided herein is an attachable reservoircontaining a TLR agonist, wherein the reservoir is attachable to adevice housing to form an ingestible device that is suitable for oraladministration to a subject and that is capable of releasing the TLRagonist at a location in the gastrointestinal tract of the subject thatis proximate to one or more sites of disease.

In particular implementation the ingestible device includes cameras(e.g., video cameras) that affords inspection of the entire GI tractwithout discomfort or the need for sedation, thus avoiding many of thepotential risks of conventional endoscopy. Video imaging can be used tohelp determine one or more characteristics of the GI tract, includingthe location of disease (e.g., presence or location of inflamed tissueand/or lesions associated with inflammatory bowel disease). In someembodiments, the ingestible device 101 may comprise a camera forgenerating video imaging data of the GI tract which can be used todetermine, among other things, the location of the device. Examples ofvideo imaging capsules include Medtronic's PillCam™, Olympus'Endocapsule®, and IntroMedic's MicroCam™. For a review of imagingcapsules, see Basar et al. “Ingestible Wireless Capsule Technology: AReview of Development and Future Indication” International Journal ofAntennas and Propagation (2012); 1-14). Other imaging technologiesimplemented with the device 101 can include thermal imaging cameras, andthose that employ ultrasound or Doppler principles to generate differentimages (see Chinese patent application CN104473611: “Capsule endoscopesystem having ultrasonic positioning function”.

Ingestible devices can be equipped with sources for generating reflectedlight, including light in the Ultraviolet, Visible, Near-infrared and/orMid-infrared spectrum, and the corresponding detectors for spectroscopyand hyperspectral imaging. Likewise, autofluorescence may be used tocharacterize GI tissue (e.g., subsurface vessel information), orlow-dose radiation (see Check-Cap™) can be used to obtain 3Dreconstructed images.

Device Components

An ingestible device in accordance with particular embodiments of thepresent invention may comprise a component made of a non-digestiblematerial and contain the TLR agonist. In some embodiments, the materialis plastic.

It is envisaged that the device is single-use. The device is loaded witha drug prior to the time of administration. In some embodiments, it maybe preferred that there is provided a medicinal product comprising thedevice pre-filled with the drug.

Anchoring Components

Several systems may actively actuate and control the capsule positionand orientation in different sections of the GI tract. Examples includeleg-like or anchor-like mechanisms that can be deployed by an ingestibledevice to resist peristaltic forces in narrowed sections of the GItract, such as the intestine, and anchor the device to a location. Othersystems employ magnetic shields of different shapes that can interactwith external magnetic fields to move the device. These mechanisms maybe particularly useful in areas outside of the small intestine, like thececum and large intestine.

An anchoring mechanism may be a mechanical mechanism. For example, adevice may be a capsule comprising a plurality of legs configured tosteer the capsule. The number of legs in the capsule may be, forexample, two, four, six, eight, ten or twelve. The aperture between thelegs of the device may be up to about 35 mm; about 30 to about 35 mm;about 35 to about 75 mm; or about 70 to about 75 mm. The contact area ofeach leg may be varied to reduce impact on the tissue. One or moremotors in the capsule may each actuate a set of legs independently fromthe other. The motors may be battery-powered motors.

An anchoring mechanism may be a non-mechanical mechanism. For example, adevice may be a capsule comprising a permanent magnet located inside thecapsule. The capsule may be anchored at the desired location of the GItract by an external magnetic field.

An anchoring mechanism may comprise a non-mechanical mechanism and amechanical mechanism. For example, a device may be a capsule comprisingone or more legs, one or more of which are coated with an adhesivematerial.

Locomotion Components

Ingestible devices can be active or passive, depending on whether theyhave controlled or non-controlled locomotion. Passive (non-controlled)locomotion is more commonly used among ingestible devices given thechallenges of implementing a locomotion module. Active (controlled)locomotion is more common in endoscopic ingestible capsules. Forexample, a capsule may comprise a miniaturized locomotion system(internal locomotion). Internal locomotion mechanisms may employindependent miniaturized propellers actuated by DC brushed motors, orthe use of water jets. As an example, a mechanism may comprise flagellaror flap-based swimming mechanisms. As an example, a mechanism maycomprise cyclic compression/extension shape-memory alloy (SMA) springactuators and anchoring systems based on directional micro-needles. Asan example, a mechanism may comprise six SMA actuated units, eachprovided with two SMA actuators for enabling bidirectional motion. As anexample, a mechanism may comprise a motor adapted to electricallystimulating the GI muscles to generate a temporary restriction in thebowel.

As an example, a capsule may comprise a magnet and motion of the capsuleis caused by an external magnetic field. For example, a locomotionsystem may comprise an ingestible capsule and an external magnetic fieldsource. For example, the system may comprise an ingestible capsule andmagnetic guidance equipment such as, for example, magnetic resonanceimaging and computer tomography, coupled to a dedicated controlinterface. In some embodiments drug release mechanisms may also betriggered by an external condition, such as temperature, pH, movement,acoustics, or combinations thereof.

Sampling Components

Ingestible devices may comprise a mechanism adapted to permit thecollection of tissue samples. In some examples, this is achieved usingelectro-mechanical solutions to collect and store the sample inside aningestible device. As an example, a biopsy mechanism may include arotational tissue cutting razor fixed to a torsional spring or the useof microgrippers to fold and collect small biopsies. As an example,Over-the-scope clips (OTSC®) may be used to perform endoscopic surgeryand/or biopsy. As an example of the methods disclosed herein, the methodmay comprise releasing a TLR agonist and collecting a sample inside thedevice. As an example, the method may comprise releasing a TLR agonistand collecting a sample inside the device in a single procedure.

FIG. 21 illustrates an example ingestible device 2100 with multipleopenings in the housing. The ingestible device 2100 has an outer housingwith a first end 2102A, a second end 2102B, and a wall 2104 extendinglongitudinally from the first end 2102A to the second end 2102B.Ingestible device 2100 has a first opening 2106 in the housing, which isconnected to a second opening 2108 in the housing. The first opening2106 of the ingestible device 2100 is oriented substantiallyperpendicular to the second opening 2108, and the connection between thefirst opening 2106 and the second opening 2108 forms a curved chamber2110 within the ingestible device 2100.

The overall shape of the ingestible device 2100, or any of the otheringestible devices discussed in this disclosure, may be similar to anelongated pill or capsule.

In some embodiments, a portion of the curved chamber 2110 may be used asa sampling chamber, which may hold samples obtained from the GI tract.In some embodiments the curved chamber 2110 is subdivided intosub-chambers, each of which may be separated by a series of one or morevalves or interlocks.

In some embodiments, the first opening 2106, the second opening 2108, orthe curved chamber 2110 include one or more of a hydrophilic orhydrophobic material, a sponge, a valve, or an air permeable membrane.

The use of a hydrophilic material or sponge may allow samples to beretained within the curved chamber 2110, and may reduce the amount ofpressure needed for fluid to enter through the first opening 2106 anddislodge air or gas in the curved chamber 2110. Examples of hydrophilicmaterials that may be incorporated into the ingestible device 2100include hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and the like. Similarly, materials that have undergonevarious types of treatments, such as plasma treatments, may havesuitable hydrophilic properties, and may be incorporated into theinvestible device 2100. Sponges may be made of any suitable material orcombination of materials, such as fibers of cotton, rayon, glass,polyester, polyethylene, polyurethane, and the like. Sponges generallymay be made from commercially available materials, such as thoseproduced by Porex®.

As discussed in more detail below, in some embodiments, the sponges maybe treated in order to change their absorbency or to help preservesamples.

In some embodiments, the sponges may be cut or abraded to change theirabsorbency or other physical properties.

Hydrophobic materials located near the second opening 2108 may repelliquids, discouraging liquid samples from entering or exiting the curvedchamber 2110 through the second opening 2108. This may serve a similarfunction as an air permeable membrane. Examples of hydrophobic materialswhich may be incorporated into the ingestible device 2100 includepolycarbonate, acrylics, fluorocarbons, styrenes, certain forms ofvinyl, stainless steel, silicone, and the like.

The various materials listed above are provided as examples, and are notlimiting. In practice, any type of suitable hydrophilic, hydrophobic, orsample preserving material may be used in the ingestible device 2100.

In some embodiments, an ingestible device includes a moveable valve as adiaphragm valve, which uses a mechanical actuator to move a flexiblediaphragm in order to seal or unseal an aperture in a second portion ofan inlet region, which may effectively block or unblock the inletregion. However, it will be understood that, in some embodiments, themoveable valve may be a different type of valve. For example, in someembodiments the moveable valve may be replaced by a pumping mechanism.As another example, in some embodiments the moveable valve is replacedwith an osmotic valve

A sampling chamber of an ingestible device can have an exit port toallow air or gas to exit the sampling chamber, while preventing at leasta portion of the sample obtained by the ingestible device from exitingthe sampling chamber. For example, the exit port may include agas-permeable membrane. An ingestible device can include one-way valveas part of its exit port.

An ingestible device can include an outlet port connected to the volumewithin housing of the ingestible device. The outlet port may provide apath for the gas to exit the ingestible device and be released into theenvironment surrounding the ingestible device. This may prevent pressurefrom building up within the housing of the ingestible device. In someembodiments, an ingestible device does not include an outlet port, andthe gas stays inside the volume of the ingestible device. In someembodiments, the outlet port may contain a gas permeable membrane, aone-way valve, a hydrophobic channel, or some other mechanism to avoidunwanted material, (e.g., fluids and solid particulates from within theGI tract), from entering the ingestible device through the outlet port.

In some embodiments, the ingestible device may include a sensor withinor proximate to the sampling chamber. For example, this sensor may beused to detect various properties of a sample contained within thesampling chamber, or this sensor may be used to detect the results of anassay technique applied to the sample contained within the samplingchamber.

In some embodiments, a hydrophilic sponge is located within the samplingchamber, and the hydrophilic sponge may be configured to absorb thesample as the sample enters the sampling chamber. In some embodiments,the hydrophilic sponge fills a substantial portion of the samplingchamber, and holds the sample for an extended period of time. This maybe particularly advantageous if the sample is collected from theingestible device after the ingestible device exits the body. In someembodiments, the hydrophilic sponge is placed on only certain surfacesor fills only certain portions of the sampling chamber. For example, itmay be possible to line certain walls (or all walls) of the samplingchamber with a hydrophilic sponge to assist in drawing in the sample,while leaving some (or none) of the walls of the sampling chamberuncovered. Leaving walls uncovered may allow the use of diagnostics orassay techniques that require a relatively un-obscured optical path.

In some embodiments, the ingestible device may include a sealed vacuumchamber connected to the exit port, or connected directly or indirectlyto the sampling chamber. In some embodiments a pin valve may be used asa moveable valve (e.g., as moveable valve of ingestible device). Incertain embodiments, a rotary valve may be used as a moveable valve(e.g., as moveable valve of ingestible device). In some embodiments, aflexible diaphragm, or diaphragm valve, may be used as a moveable valve(e.g., as moveable valve of ingestible device). In certain embodiments,a mechanism is near the diaphragm or in direct contact with thediaphragm. The spring mechanism may apply pressure to the diaphragm tooppose the pressure applied by the mechanical actuator, which may causethe flexible diaphragm to be moved into an open position when themechanical actuator is not applying pressure to the flexible diaphragm.Additionally, this may ensure that the diaphragm valve remains open whenthe mechanical actuator is not applying pressure across the flexiblediaphragm. In some embodiments, moving the mechanical actuator from aclosed position to an open position causes a volume of the inlet regionwithin the ingestible device to increase. This may cause the pressurewithin the inlet region to be reduced, generating suction to draw asample into the inlet region. Similarly, moving the mechanical actuatorfrom an open position to a closed position may cause the volume of theinlet region to be reduced. This may cause the pressure within the inletregion to be increased, pushing the sample out of the inlet region.Depending on the design of the inlet region, the mechanical actuator,and the moveable valve, this may push the sample into the samplingchamber rather than pushing the sample back through the opening in theingestible device.

FIG. 22 depicts a cross-sectional view of a portion of the interior ofingestible device 3000. As shown in FIG. 22, the interior of ingestibledevice 3000 includes a valve system 3100 and a sampling system 3200.Valve system 3100 is depicted as having a portion that is flush with theopening 3018 so that valve system 3100 prevents fluid exterior toingestible device 2000 from entering sampling system 3200. However, asdescribed in more detail below with reference to FIGS. 22-27, valvesystem 3100 can change position so that valve system 3100 allows fluidexterior to ingestible device 3000 to enter sampling system 3200.

FIGS. 23 and 27 illustrate valve system 3100 in more detail. As shown inFIG. 23, valve system 3100 includes an actuation mechanism 3110, atrigger 3120, and a gate 3130. In FIGS. 23 and 7, a leg 3132 of gate3130 is flush against, and parallel with, housing wall 3016 so that gateleg 3132 covers opening 3018 to prevent fluid exterior to ingestibledevice 3000 (e.g., fluid in the GI tract) from entering the interior ofingestible device 3000. A protrusion 3134 of gate 3130 engages a lip3122 of trigger 3120. A peg 3124 of trigger 3120 engages a wax pot 3112of actuation mechanism 3110. Referring to FIG. 27, a biasing mechanism3140 includes a compression spring 3142 that applies an upward force ongate 3130. Biasing mechanism 3140 also includes a torsion spring 3144that applies a force on trigger 3120 in the counter-clockwise direction.In FIGS. 23 and 27, the force applied by torsion spring 3144 iscounter-acted by the solid wax in pot 3112, and the force applied bycompression spring 3142 is counter-acted by lip 3122.

FIG. 24A and FIG. 24B show an embodiment of the manner in whichactuation mechanism 3110 actuates movement of trigger 3120. Similar toFIGS. 23 and 27, FIG. 24A shows a configuration in which peg 3124applies a force against solid wax pot 3112 due to torsion spring 3144,and in which the solid nature of wax pot 3112 resists the force appliedby peg 3124. A control unit 3150 is in signal communication with valvesystem 3100. During use of ingestible device 3000, a control unit 3150receives a signal, indicating that the position of valve system 3100should change, e.g., so that ingestible device 3000 can take a sample ofa fluid in the GI tract. Control unit 3150 sends a signal that causes aheating system 3114 of actuation system 3100 to heat the wax in pot 3112so that the wax melts. As shown in FIG. 24B, the melted wax is not ableto resist the force applied by peg 3124 so that, under the force oftorsion spring 3144, trigger 3120 moves in a counter-clockwise fashion.

FIGS. 25A and 25B illustrate the interaction of trigger 3120 and gate3130 before and after actuation. As shown in FIG. 25A, when wax pot 3112is solid (corresponding to the configuration shown in FIG. 24A),protrusion 3134 engages lip 3122, which prevents the force ofcompression spring 3142 from moving gate 3130 upward. As shown in FIG.25B, when the wax in pot 3112 melts (FIG. 24B), trigger 3120 movescounter-clockwise, and lip 3122 disengages from protrusion 3134. Thisallows the force of compression spring 3142 to move gate 3130 upward. Asseen by comparing FIG. 25A to FIG. 25B, the upward movement of gate 3130results in an upward movement of an opening 3136 in gate leg 3132.

FIGS. 26A and 26B illustrate the impact of the upward movement ofopening 3136 on the ability of ingestible device 3000 to obtain asample. As shown in FIG. 26A, when the wax in pot 3112 is solid (FIGS.24A and 25A), opening 3136 in is not aligned with opening 3018 in wall3016 of ingestible device 3000. Instead, gate leg 3132 covers opening3018 and blocks fluid from entering the interior of ingestible device3000. As shown in FIG. 26B, when the wax in pot 3112 is melted andtrigger 3120 and gate 3130 have moved (FIGS. 24B and 42B), opening 3136in gate 3130 is aligned with opening 3018 in wall 3016. In thisconfiguration, fluid that is exterior to ingestible device 3000 (e.g.,in the GI tract) can enter the interior of ingestible device 3000 viaopenings 3018 and 3036.

FIG. 27 illustrates a more detailed view of ingestible device 3000including valve system 3100 and sampling system 3200.

While the foregoing description is made with regard to a valve systemhaving one open position and one closed position (e.g., a two-stagevalve system), the disclosure is not limited in this sense. Rather, theconcepts described above with regard to a two stage valve system can beimplemented with a valve system have more than two stages (e.g., threestages, four stages, five stages, etc.).

As noted above in addition to a valve system, an ingestible deviceincludes a sampling system. FIG. 28 illustrates a partial crosssectional view of ingestible device 3000 with sampling system 3200 andcertain components of valve system 3100. Sampling system 3200 includes aseries of sponges configured to absorb fluid from an opening, move thefluid to a location within the housing, and prepare the fluid fortesting. Preparation for testing may include filtering the fluid andcombining the fluid with a chemical assay. The assay may be configuredto dye cells in the filtered sample. The series of sponges includes awicking sponge 3210, a transfer sponge 3220, a volume sponge 3230, andan assay sponge 3240. Sampling system 3200 also includes a membrane 3270located between assay sponge 3240 and a vent 3280 for gases to leavesampling system 3200. A cell filter 3250 is located between distal end3214 of wicking sponge 3210 and a first end 3222 of transfer sponge3220. Membrane 3270 is configured to allow one or more gases to leavesampling system 3200 via an opening 3280, while maintaining liquid insampling system 3200.

FIG. 29 is a highly schematic illustration of an ingestible device 4000that contains multiple different systems that cooperate for obtaining asample and analyzing a sample, e.g., within the GI tract of a subject.Ingestible device 4000 includes a power system 4100 (e.g., one or morebatteries), configured to power an electronics system 4200 (e.g.,including a control system, optionally in signal communication with anexternal base station), a valve system 4300, a sampling system 4400, andan analytic system 4500. Exemplary analytical systems include assaysystems, such as, for example, optical systems containing one or moresources of radiation and/or one more detectors.

Some or all of the sponges of the above-described sampling systems maycontain one or more preservatives (see discussion above). Typically, theassay sponge and/or the volume sponge 3230 and/or the transfer spongecontain one or more preservatives. Typically, the preservative(s) areselected based on the analyte of interest, e.g., an analyte (such as aprotein biomarker) for a GI disorder.

Communication Systems

An ingestible device may be equipped with a communication system adaptedto transmit and/or receive data, including imaging and/or localizationdata. As an example, a communication system may employ radiofrequencytransmission. Ingestible devices using radiofrequency communication areattractive because of their efficient transmission through the layers ofthe skin. This is especially true for low frequency transmission(UHF-433 ISM and lower, including the Medical Device Radio CommunicationService band (MDRS) band 402-406 MHz). In another embodiment, acousticsare used for communications, including the transmission of data. Forexample, an ingestible capsule may be able to transmit information byapplying one or more base voltages to an electromechanical transducer orpiezoelectric (e.g., PZT, PVDF, etc.) device to cause the piezoelectricdevice to ring at particular frequencies, resulting in an acoustictransmission. A multi-sensor array for receiving the acoustictransmission may include a plurality of acoustic transducers thatreceive the acoustic transmission from a movable device such as aningestible capsule as described in U.S. patent application Ser. No.11/851,214 filed Sep. 6, 2007, incorporated by reference herein in itsentirety.

As an example, a communication system may employ human bodycommunication technology. Human body communication technology uses thehuman body as a conductive medium, which generally requires a largenumber of sensor electrodes on the skin. As an example, a communicationsystem may integrate a data storage system.

Environmental Sensors

In some embodiments the device may comprise environmental sensors tomeasure pH, temperature, transit times, or combinations thereof. Otherexamples of environmental sensors include, but are not limited to acapacitance sensor, an impedance sensor, a heart rate sensor, acousticsensor such as a microphone or hydrophone, image sensor, and/or amovement sensor. In one embodiment, the ingestible device comprises aplurality of different environmental sensors for generating differentkinds of environmental data.

In order to avoid the problem of capsule retention, a thorough pastmedical and surgical history should be undertaken. In addition, severalother steps have been proposed, including performing investigations suchas barium follow-through. In cases where it is suspected that there is ahigh risk of retention, the patient is given a patency capsule a fewdays before swallowing an ingestible device. Any dissolvablenon-endoscopic capsule may be used to determine the patency of the GItract. The patency capsule is usually the same size as the ingestibledevice and can be made of cellophane. In some embodiments, the patencycapsule contains a mixture of barium and lactose, which allowsvisualization by x-ray. The patency capsule may also include a radiotagor other label, which allows for it to be detected by radio-scannerexternally. The patency capsule may comprise wax plugs, which allow forintestinal fluid to enter and dissolve the content, thereby dividing thecapsule into small particles.

Accordingly, in some embodiments, the methods herein comprise (a)identifying a subject having a disease of the gastrointestinal tract and(b) evaluating the subject for suitability to treatment. In someembodiments, the methods herein comprise evaluating for suitability totreatment a subject identified as having a disease of thegastrointestinal tract. In some embodiments, evaluating the subject forsuitability to treatment comprises determining the patency of thesubject's GI tract.

In some embodiments, an ingestible device comprises a tissue anchoringmechanism for anchoring the ingestible device to a subject's tissue. Forexample, an ingestible device could be administered to a subject andonce it reaches the desired location, the tissue attachment mechanismcan be activated or deployed such that the ingestible device, or aportion thereof, is anchored to the desired location. In someembodiments, the tissue anchoring mechanism is reversible such thatafter initial anchoring, the tissue attachment device is retracted,dissolved, detached, inactivated or otherwise rendered incapable ofanchoring the ingestible device to the subject's tissue. In someembodiments the attachment mechanism is placed endoscopically.

In some embodiments, a tissue anchoring mechanism comprises anosmotically-driven sucker. In some embodiments, the osmotically-drivensucker comprises a first valve on the near side of theosmotically-driven sucker (e.g., near the subject's tissue) and a secondone-way valve that is opened by osmotic pressure on the far side of theosmotically-driven sucker, and an internal osmotic pump systemcomprising salt crystals and semi-permeable membranes positioned betweenthe two valves. In such embodiments, osmotic pressure is used to adherethe ingestible device to the subject's tissue without generating avacuum within the ingestible capsule. After the osmotic system isactivated by opening the first valve, fluid is drawn in through thesucker and expelled through the second burst valve. Fluid continues toflow until all the salt contained in the sucker is dissolved or untiltissue is drawn into the sucker. As liminal fluid is drawn through theosmotic pump system, solutes build up between the tissue and the firstvalve, reducing osmotic pressure. In some embodiments, the solutebuildup stalls the pump before the tissue contacts the valve, preventingtissue damage. In some embodiments, a burst valve is used on the farside of the osmotically-driven sucker rather than a one-way valve, suchthat luminal fluid eventually clears the saline chamber and the osmoticflow reverses, actively pushing the subject's tissue out of the sucker.In some embodiments, the ingestible device may be anchored to theinterior surface of tissues forming the GI tract of a subject. In oneembodiment, the ingestible device comprises a connector for anchoringthe device to the interior surface of the GI tract. The connector may beoperable to ingestible device to the interior surface of the GI tractusing an adhesive, negative pressure and/or fastener.

In some embodiments a device comprises a tract stimulator and/or monitorIMD comprising a housing enclosing electrical stimulation and/ormonitoring circuitry and a power source and an elongated flexible memberextending from the housing to an active fixation mechanism adapted to befixed into the GI tract wall is disclosed. After fixation is effected,the elongated flexible member bends into a preformed shape that pressesthe housing against the mucosa so that forces that would tend todislodge the fixation mechanism are minimized. The IMD is fitted into anesophageal catheter lumen with the fixation mechanism aimed toward thecatheter distal end opening whereby the bend in the flexible member isstraightened. The catheter body is inserted through the esophagus intothe GI tract cavity to direct the catheter distal end to the site ofimplantation and fix the fixation mechanism to the GI tract wall. TheIMD is ejected from the lumen, and the flexible member assumes its bentconfiguration and lodges the hermetically sealed housing against themucosa. A first stimulation/sense electrode is preferably an exposedconductive portion of the housing that is aligned with the bend of theflexible member so that it is pressed against the mucosa. A secondstimulation/sense electrode is located at the fixation site.

In some embodiments a device includes a fixation mechanism to anchor thedevice to tissue within a body lumen, and a mechanism to permitselective de-anchoring of the device from the tissue anchoring sitewithout the need for endoscopic or surgical intervention. Anelectromagnetic device may be provided to mechanically actuate thede-anchoring mechanism. Alternatively, a fuse link may be electricallyblown to de-anchor the device. As a further alternative, a rapidlydegradable bonding agent may be exposed to a degradation agent tode-anchor the device from a bonding surface within the body lumen.

In some embodiments a device is as disclosed in patent publicationWO2015112575A1, incorporated by reference herein in its entirety. Thepatent publication is directed to a gastrointestinal sensor implantationsystem. In some embodiments an orally-administrable capsule comprises atissue capture device or reservoir removably coupled to theorally-administrable capsule, where the tissue capture device includinga plurality of fasteners for anchoring the tissue capture device togastrointestinal tissue within a body

In some embodiments, the ingestible device contains an electric energyemitting means, a radio signal transmitting means, a medicament storagemeans and a remote actuatable medicament releasing means. The capsulesignals a remote receiver as it progresses through the alimentary tractin a previously mapped route and upon reaching a specified site isremotely triggered to release a dosage of medicament. Accordingly, insome embodiments, releasing the TLR agonist is triggered by a remoteelectromagnetic signal.

In some embodiments, the ingestible device includes a housingintroducible into a body cavity and of a material insoluble in the bodycavity fluids, but formed with an opening covered by a material which issoluble in body cavity fluids. A diaphragm divides the interior of thehousing into a medication chamber including the opening, and a controlchamber. An electrolytic cell in the control chamber generates a gaswhen electrical current is passed therethrough to deliver medicationfrom the medication chamber through the opening into the body cavity ata rate controlled by the electrical current. Accordingly, in someembodiments, releasing the TLR agonist is triggered by generation in thecomposition of a gas in an amount sufficient to expel the TLR agonist.

In some embodiments, the ingestible device includes an oral drugdelivery device having a housing with walls of water permeable materialand having at least two chambers separated by a displaceable membrane.The first chamber receives drug and has an orifice through which thedrug is expelled under pressure. The second chamber contains at leastone of two spaced apart electrodes forming part of an electric circuitwhich is closed by the ingress of an aqueous ionic solution into thesecond chamber. When current flows through the circuit, gas is generatedand acts on the displaceable membrane to compress the first chamber andexpel the active ingredient through the orifice for progressive deliveryto the gastrointestinal tract.

In some embodiments, the ingestible device includes an ingestible devicefor delivering a substance to a chosen location in the GI tract of amammal includes a receiver of electromagnetic radiation for powering anopenable part of the device to an opened position for dispensing of thesubstance. The receiver includes a coiled wire that couples the energyfield, the wire having an air or ferrite core. In a further embodimentthe invention includes an apparatus for generating the electromagneticradiation, the apparatus including one or more pairs of field coilssupported in a housing. The device optionally includes a latch definedby a heating resistor and a fusible restraint. The device may alsoinclude a flexible member that may serve one or both the functions ofactivating a transmitter circuit to indicate dispensing of thesubstance; and restraining of a piston used for expelling the substance.

In some embodiments, the ingestible device includes an ingestible devicefor delivering a substance to a chosen location in the GI tract of amammal includes a receiver of electromagnetic radiation for powering anopenable part of the device to an opened position for dispensing of thesubstance. The receiver includes a coiled wire that couples the energyfield, the wire having an air or ferrite core. In a further embodimentthe invention includes an apparatus for generating the electromagneticradiation, the apparatus including one or more pairs of field coilssupported in a housing. The device optionally includes a latch definedby a heating resistor and a fusible restraint. The device may alsoinclude a flexible member that may serve one or both the functions ofactivating a transmitter circuit to indicate dispensing of thesubstance; and restraining of a piston used for expelling the substance.

In some embodiments, the ingestible device is a device a swallowablecapsule. A sensing module is disposed in the capsule. A bioactivesubstance dispenser is disposed in the capsule. A memory and logiccomponent is disposed in the capsule and in communication with thesensing module and the dispenser.

In some embodiments, localized administration is implemented via anelectronic probe which is introduced into the intestinal tract of aliving organism and which operates autonomously therein, adapted todeliver one or more therapy agents. In one embodiment, the methodincludes loading the probe with one or more therapy agents, andselectively releasing the agents from the probe at a desired location ofthe intestinal tract in order to provide increased efficacy overtraditional oral ingestion or intravenous introduction of the agent(s).

In some embodiments, the ingestible device includes electronic controlmeans for dispensing the drug substantially to the diseased tissue sitesof the GI tract, according to a pre-determined drug release profileobtained prior to administration from the specific mammal. Accordingly,in some embodiments, releasing the TLR agonist is triggered by anelectromagnetic signal generated within the device. The releasing mayoccur according to a pre-determined drug release profile.

In some embodiments, the ingestible device can include at least oneguide tube, one or more tissue penetrating members positioned in theguide tube, a delivery member, an actuating mechanism and a releaseelement. The release element degrades upon exposure to variousconditions in the intestine so as to release and actuate the actuatingmechanism. Embodiments of the invention are particularly useful for thedelivery of drugs which are poorly absorbed, tolerated and/or degradedwithin the GI tract.

In some embodiments, the ingestible device includes an electronic pillcomprising at least one reservoir with a solid powder or granulatemedicament or formulation, a discharge opening and an actuatorresponsive to control circuitry for displacing medicine from thereservoir to the discharge opening. The medicament or formulationcomprises a dispersion of one or more active ingredients—e.g., solids inpowder or granulate form—in an inert carrier matrix. Optionally, theactive ingredients are dispersed using intestinal moisture absorbed intothe pill via a semi-permeable wall section.

In some embodiments, the ingestible device includes a sensor comprisinga plurality of electrodes having a miniature size and a lower powerconsumption and a coating exterior to the electrodes, wherein thecoating interacts with a target condition thereby producing a change inan electrical property of the electrodes, wherein the change istransduced into an electrical signal by the electrodes. Accordingly, insome embodiments, releasing the TLR agonist is triggered by an electricsignal by the electrodes resulting from the interaction of the coatingwith the one or more sites of disease. Further provided herein is asystem for medication delivery comprising such sensor and a pill.

In some embodiments, the ingestible device includes an electronic pillcomprising a plurality of reservoirs, each of the reservoirs comprisinga discharge opening covered by a removable cover. The pill comprises atleast one actuator responsive to control circuitry for removing thecover from the discharge opening. The actuator can for example be aspring loaded piston breaking a foil cover when dispensing themedicament. Alternatively, the cover can be a rotatable disk or cylinderwith an opening which can be brought in line with the discharge openingof a reservoir under the action of the actuator.

In some embodiments, the ingestible device includes an electronicallyand remotely controlled pill or medicament delivery system. The pillincludes a housing; a reservoir for storing a medicament; anelectronically controlled release valve or hatch for dispensing one ormore medicaments stored in the reservoir while traversing thegastrointestinal tract; control and timing circuitry for opening andclosing the valve; and a battery. The control and timing circuitry opensand closes the valve throughout a dispensing time period in accordancewith a preset dispensing timing pattern which is programmed within thecontrol and timing circuitry. RF communication circuitry receivescontrol signals for remotely overriding the preset dispensing timingpattern, reprogramming the control and timing circuitry or terminatingthe dispensing of the medicament within the body. The pill includes anRFID tag for tracking, identification, inventory and other purposes.

In some embodiments, the ingestible device includes an electroniccapsule which has a discrete drive element comprising: a housing,electronics for making the electronic capsule operable, a pumpingmechanism for dosing and displacing a substance, a power source forpowering the electronic capsule and enabling the electronics and thepumping mechanism to operate, and a locking mechanism; and a discretepayload element comprising: a housing, a reservoir for storing thesubstance, one or more openings in the housing for releasing thesubstance from the reservoir and a locking mechanism for engaging thedrive element locking mechanism. Engagement of the drive element lockingmechanism with the payload element locking mechanism secures the driveelement to the payload element, thereby making the electronic capsuleoperable and specific.

In some embodiments, the ingestible device may be a mucoadhesive deviceconfigured for release of an active agent.

In some embodiments, the ingestible device includes an apparatus thatincludes an ingestible medical treatment device, which is configured toinitially assume a contracted state having a volume of less than 4 cm³.The device includes a gastric anchor, which initially assumes acontracted size, and which is configured to, upon coming in contact witha liquid, expand sufficiently to prevent passage of the anchor through around opening having a diameter of between 1 cm and 3 cm. The devicealso includes a duodenal unit, which is configured to pass through theopening, and which is coupled to the gastric anchor such that theduodenal unit is held between 1 cm and 20 cm from the gastric anchor.

In some embodiments, the ingestible device includes a medical roboticsystem and method of operating such comprises taking intraoperativeexternal image data of a patient anatomy, and using that image data togenerate a modeling adjustment for a control system of the medicalrobotic system (e.g., updating anatomic model and/or refining instrumentregistration), and/or adjust a procedure control aspect (e.g.,regulating substance or therapy delivery, improving targeting, and/ortracking performance).

In one embodiment the ingestible device may also include one or moreenvironmental sensors. Environmental sensor may be used to generateenvironmental data for the environment external to device in thegastrointestinal (GI) tract of the subject. In some embodiments,environmental data is generated at or near the location within the GItract of the subject where a drug is delivered. Examples ofenvironmental sensor include, but are not limited to a capacitancesensor, a temperature sensor, an impedance sensor, a pH sensor, a heartrate sensor, acoustic sensor, image sensor (e.g., a hydrophone), and/ora movement sensor (e.g., an accelerometer). In one embodiment, theingestible device comprises a plurality of different environmentalsensors for generating different kinds of environmental data.

In one embodiment, the image sensor is a video camera suitable forobtaining images in vivo of the tissues forming the GI tract of thesubject. In one embodiment, the environmental data is used to helpdetermine one or more characteristics of the GI tract, including thelocation of disease (e.g., presence or location of inflamed tissueand/or lesions associated with inflammatory bowel disease). In someembodiments, the ingestible device may comprise a camera for generatingvideo imaging data of the GI tract which can be used to determine, amongother things, the location of the device.

In another embodiment, the ingestible device described herein may belocalized using a gamma scintigraphy technique or other radio-trackertechnology as employed by Phaeton Research's Enterion™ capsule (SeeTeng, Renli, and Juan Maya. “Absolute bioavailability and regionalabsorption of ticagrelor in healthy volunteers.” Journal of DrugAssessment 3.1 (2014): 43-50), or monitoring the magnetic field strengthof permanent magnet in the ingestible device (see T. D. Than, et al., “Areview of localization systems for robotic endoscopic capsules,” IEEETrans. Biomed. Eng., vol. 59, no. 9, pp. 2387-2399, September 2012).

In one embodiment, drug delivery is triggered when it encounters thesite of disease in the GI tract.

In one embodiment, the one or more environmental sensors measure pH,temperature, transit times, or combinations thereof.

In some embodiments, releasing the TLR agonist is dependent on the pH ator in the vicinity of the location. In some embodiments the pH in thejejunum is from 6.1 to 7.2, such as 6.6. In some embodiments the pH inthe mid small bowel is from 7.0 to 7.8, such as 7.4. In some embodimentsthe pH in the ileum is from 7.0 to 8.0, such as 7.5. In some embodimentsthe pH in the right colon is from 5.7 to 7.0, such as 6.4. In someembodiments the pH in the mid colon is from 5.7 to 7.4, such as 6.6. Insome embodiments the pH in the left colon is from 6.3 to 7.7, such as7.0. In some embodiments, the gastric pH in fasting subjects is fromabout 1.1 to 2.1, such as from 1.4 to 2.1, such as from 1.1 to 1.6, suchas from 1.4 to 1.6. In some embodiments, the gastric pH in fed subjectsis from 3.9 to 7.0, such as from 3.9 to 6.7, such as from 3.9 to 6.4,such as from 3.9 to 5.8, such as from 3.9 to 5.5, such as from 3.9 to5.4, such as from 4.3 to 7.0, such as from 4.3 to 6.7, such as from 4.3to 6.4, such as from 4.3 to 5.8, such as from 4.3 to 5.5, such as from4.3 to 5.4. In some embodiments, the pH in the duodenum is from 5.8 to6.8, such as from 6.0 to 6.8, such as from 6.1 to 6.8, such as from 6.2to 6.8, such as from 5.8 to 6.7, such as from 6.0 to 6.7, such as from6.1 to 6.7, such as from 6.2 to 6.7, such as from 5.8 to 6.6, such asfrom 6.0 to 6.6, such as from 6.1 to 6.6, such as from 6.2 to 6.6, suchas from 5.8 to 6.5, such as from 6.0 to 6.5, such as from 6.1 to 6.5,such as from 6.2 to 6.5.

In some embodiments, releasing the TLR agonist is not dependent on thepH at or in the vicinity of the location. In some embodiments, releasingthe TLR agonist is triggered by degradation of a release componentlocated in the capsule. In some embodiments, the TLR agonist is nottriggered by degradation of a release component located in the capsule.In some embodiments, wherein releasing the TLR agonist is not dependenton enzymatic activity at or in the vicinity of the location. In someembodiments, releasing the TLR agonist is not dependent on bacterialactivity at or in the vicinity of the location.

In some embodiments, the pharmaceutical composition is an ingestibledevice, comprising:

a housing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from the firstend to the second end;

a reservoir located within the housing and containing the TLR agonist,wherein a first end of the reservoir is attached to the first end of thehousing;

a mechanism for releasing the TLR agonist from the reservoir;

and;

an exit valve configured to allow the TLR agonist to be released out ofthe housing from the reservoir.

In some embodiments, the ingestible device further comprises:

an electronic component located within the housing; and

a gas generating cell located within the housing and adjacent to theelectronic component,

wherein the electronic component is configured to activate the gasgenerating cell to generate gas.

In some embodiments, the ingestible device further comprises:

a safety device placed within or attached to the housing,

-   -   wherein the safety device is configured to relieve an internal        pressure within the housing when the internal pressure exceeds a        threshold level.

In some embodiments, the pharmaceutical composition is an ingestibledevice, comprising:

a housing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from the firstend to the second end;

an electronic component located within the housing;

a gas generating cell located within the housing and adjacent to theelectronic component,

-   -   wherein the electronic component is configured to activate the        gas generating cell to generate gas;

a reservoir located within the housing,

-   -   wherein the reservoir stores a dispensable substance and a first        end of the reservoir is attached to the first end of the        housing;

an exit valve located at the first end of the housing,

-   -   wherein the exit valve is configured to allow the dispensable        substance to be released out of the first end of the housing        from the reservoir; and

a safety device placed within or attached to the housing,

-   -   wherein the safety device is configured to relieve an internal        pressure within the housing when the internal pressure exceeds a        threshold level.

In some embodiments, the pharmaceutical composition is an ingestibledevice, comprising:

a housing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from the firstend to the second end;

an electronic component located within the housing,

a gas generating cell located within the housing and adjacent to theelectronic component,

-   -   wherein the electronic component is configured to activate the        gas generating cell to generate gas;

a reservoir located within the housing,

-   -   wherein the reservoir stores a dispensable substance and a first        end of the reservoir is attached to the first end of the        housing;

an injection device located at the first end of the housing,

-   -   wherein the jet injection device is configured to inject the        dispensable substance out of the housing from the reservoir; and

a safety device placed within or attached to the housing,

-   -   wherein the safety device is configured to relieve an internal        pressure within the housing.

In some embodiments, the pharmaceutical composition is an ingestibledevice, comprising:

a housing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from the firstend to the second end;

an optical sensing unit located on a side of the housing,

-   -   wherein the optical sensing unit is configured to detect a        reflectance from an environment external to the housing;

an electronic component located within the housing;

a gas generating cell located within the housing and adjacent to theelectronic component,

-   -   wherein the electronic component is configured to activate the        gas generating cell to generate gas in response to identifying a        location of the ingestible device based on the reflectance;

a reservoir located within the housing,

-   -   wherein the reservoir stores a dispensable substance and a first        end of the reservoir is attached to the first end of the        housing;

a membrane in contact with the gas generating cell and configured tomove or deform into the reservoir by a pressure generated by the gasgenerating cell; and

a dispensing outlet placed at the first end of the housing,

-   -   wherein the dispensing outlet is configured to deliver the        dispensable substance out of the housing from the reservoir.

In one embodiment, drug delivery is triggered when it encounters thesite of disease in the GI tract.

In one embodiment, the one or more environmental sensors measure pH,temperature, transit times, or combinations thereof.

In some embodiments, releasing the TLR agonist is dependent on the pH ator in the vicinity of the location. In some embodiments the pH in thejejunum is from 6.1 to 7.2, such as 6.6. In some embodiments the pH inthe mid small bowel is from 7.0 to 7.8, such as 7.4. In some embodimentsthe pH in the ileum is from 7.0 to 8.0, such as 7.5. In some embodimentsthe pH in the right colon is from 5.7 to 7.0, such as 6.4. In someembodiments the pH in the mid colon is from 5.7 to 7.4, such as 6.6. Insome embodiments the pH in the left colon is from 6.3 to 7.7, such as7.0. In some embodiments, the gastric pH in fasting subjects is fromabout 1.1 to 2.1, such as from 1.4 to 2.1, such as from 1.1 to 1.6, suchas from 1.4 to 1.6. In some embodiments, the gastric pH in fed subjectsis from 3.9 to 7.0, such as from 3.9 to 6.7, such as from 3.9 to 6.4,such as from 3.9 to 5.8, such as from 3.9 to 5.5, such as from 3.9 to5.4, such as from 4.3 to 7.0, such as from 4.3 to 6.7, such as from 4.3to 6.4, such as from 4.3 to 5.8, such as from 4.3 to 5.5, such as from4.3 to 5.4. In some embodiments, the pH in the duodenum is from 5.8 to6.8, such as from 6.0 to 6.8, such as from 6.1 to 6.8, such as from 6.2to 6.8, such as from 5.8 to 6.7, such as from 6.0 to 6.7, such as from6.1 to 6.7, such as from 6.2 to 6.7, such as from 5.8 to 6.6, such asfrom 6.0 to 6.6, such as from 6.1 to 6.6, such as from 6.2 to 6.6, suchas from 5.8 to 6.5, such as from 6.0 to 6.5, such as from 6.1 to 6.5,such as from 6.2 to 6.5.

In some embodiments, releasing the TLR agonist is not dependent on thepH at or in the vicinity of the location. In some embodiments, releasingthe TLR agonist is triggered by degradation of a release componentlocated in the capsule. In some embodiments, the TLR agonist is nottriggered by degradation of a release component located in the capsule.In some embodiments, wherein releasing the TLR agonist is not dependenton enzymatic activity at or in the vicinity of the location. In someembodiments, releasing the TLR agonist is not dependent on bacterialactivity at or in the vicinity of the location.

In some embodiments, the pharmaceutical composition is an ingestibledevice, comprising:

a housing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from the firstend to the second end;

a reservoir located within the housing and containing the TLR agonist,

wherein a first end of the reservoir is attached to the first end of thehousing;

a mechanism for releasing the TLR agonist from the reservoir;

and;

an exit valve configured to allow the TLR agonist to be released out ofthe housing from the reservoir.

In some embodiments, the ingestible device further comprises:

an electronic component located within the housing; and

a gas generating cell located within the housing and adjacent to theelectronic component,

wherein the electronic component is configured to activate the gasgenerating cell to generate gas.

In some embodiments, the ingestible device further comprises:

a safety device placed within or attached to the housing,

-   -   wherein the safety device is configured to relieve an internal        pressure within the housing when the internal pressure exceeds a        threshold level.

In some embodiments, the pharmaceutical composition is an ingestibledevice, comprising:

a housing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from the firstend to the second end;

an electronic component located within the housing;

a gas generating cell located within the housing and adjacent to theelectronic component,

-   -   wherein the electronic component is configured to activate the        gas generating cell to generate gas;

a reservoir located within the housing,

-   -   wherein the reservoir stores a dispensable substance and a first        end of the reservoir is attached to the first end of the        housing;

an exit valve located at the first end of the housing,

-   -   wherein the exit valve is configured to allow the dispensable        substance to be released out of the first end of the housing        from the reservoir; and

a safety device placed within or attached to the housing,

-   -   wherein the safety device is configured to relieve an internal        pressure within the housing when the internal pressure exceeds a        threshold level.

In some embodiments, the pharmaceutical composition is an ingestibledevice, comprising:

a housing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from the firstend to the second end;

an electronic component located within the housing,

a gas generating cell located within the housing and adjacent to theelectronic component,

-   -   wherein the electronic component is configured to activate the        gas generating cell to generate gas;

a reservoir located within the housing,

-   -   wherein the reservoir stores a dispensable substance and a first        end of the reservoir is attached to the first end of the        housing;

an injection device located at the first end of the housing,

-   -   wherein the jet injection device is configured to inject the        dispensable substance out of the housing from the reservoir; and

a safety device placed within or attached to the housing,

-   -   wherein the safety device is configured to relieve an internal        pressure within the housing.

In some embodiments, the pharmaceutical composition is an ingestibledevice, comprising:

a housing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from the firstend to the second end;

an optical sensing unit located on a side of the housing,

-   -   wherein the optical sensing unit is configured to detect a        reflectance from an environment external to the housing;

an electronic component located within the housing;

a gas generating cell located within the housing and adjacent to theelectronic component,

-   -   wherein the electronic component is configured to activate the        gas generating cell to generate gas in response to identifying a        location of the ingestible device based on the reflectance;

a reservoir located within the housing,

-   -   wherein the reservoir stores a dispensable substance and a first        end of the reservoir is attached to the first end of the        housing;

a membrane in contact with the gas generating cell and configured tomove or deform into the reservoir by a pressure generated by the gasgenerating cell; and

a dispensing outlet placed at the first end of the housing,

-   -   wherein the dispensing outlet is configured to deliver the        dispensable substance out of the housing from the reservoir.

In some embodiments, the pharmaceutical composition is an ingestibledevice as disclosed in U.S. Patent Application Ser. No. 62/385,553,incorporated by reference herein in its entirety.

In some embodiments, the pharmaceutical composition is an ingestibledevice as disclosed in the following applications, each of which isincorporated by reference herein in its entirety:

U.S. Ser. Nos. 14/460,893; 15/514,413; 62/376,688; 62/385,344;62/478,955; 62/434,188; 62/434,320; 62/431,297; 62/434,797; 62/480,187;62/502,383; and 62/540,873.

In some embodiments, the pharmaceutical composition is an ingestibledevice comprising a localization mechanism as disclosed in internationalpatent application PCT/US2015/052500, incorporated by reference hereinin its entirety.

In some embodiments, the pharmaceutical composition is not a dart-likedosage form.

In some embodiments of any ingestible device disclosed herein comprisinga TLR agonist, the TLR agonist is present in a therapeutically effectiveamount.

In case of conflict between the present specification and any subjectmatter incorporated by reference herein, the present specification,including definitions, will control.

Devices and Methods for Detection of Analytes in GI Tract

Detection of certain analytes in the GI tract may be useful in theidentification of the nature and severity of the disease, in accuratelylocating the site(s) of disease, and in assessing patient response to atherapeutic agent. The appropriate therapeutic agent may accordingly bereleased at the correct locations(s), dosage, or timing for the disease.As discussed further herein, analytes may include biomarkers associatedwith a disease or associated with patient response and/or therapeuticagents previously administered to treat the disease. In someembodiments, the disclosure provides an ingestible device for detectingan analyte in a sample, the ingestible device comprising a samplingchamber that is configured to hold a composition comprising: (1) aplurality of donor particles, each of the plurality of donor particlescomprising a photosensitizer and having coupled thereto a firstantigen-binding agent that binds to the analyte, wherein thephotosensitizer, in its excited state, is capable of generating singletoxygen; and (2) a plurality of acceptor particles, each of the pluralityof acceptor particles comprising a chemiluminescent compound and havingcoupled thereto a second antigen-binding agent that binds to theanalyte, wherein the chemiluminescent compound is capable of reactingwith singlet oxygen to emit luminescence. In some embodiments, the firstand the second analyte-binding agents are antigen-binding agents (e.g.,antibodies). In some embodiments, the first and the secondantigen-binding agents bind to the same epitope of the analyte (e.g., aprotein). In some embodiments, the first and the second antigen-bindingagents bind to separate epitopes of the analyte (e.g., a protein) thatspatially overlap. In some embodiments, the first and the secondantigen-binding agents bind to the separate epitopes of the analyte(e.g., a protein) that do not spatially overlap.

In some embodiments, this disclosure provides an ingestible device fordetecting an analyte in a sample, the ingestible device comprising asampling chamber that is configured to hold an absorbable material(e.g., an absorbable pad or sponge) having absorbed therein acomposition comprising: (1) a plurality of donor particles, each of theplurality of donor particles comprising a photosensitizer and havingcoupled thereto a first antigen-binding agent that binds to the analyte,wherein the photosensitizer, in its excited state, is capable ofgenerating singlet oxygen; and (2) a plurality of acceptor particles,each of the plurality of acceptor particles comprising achemiluminescent compound and having coupled thereto a secondantigen-binding agent that binds to the analyte, wherein thechemiluminescent compound is capable of reacting with singlet oxygen toemit luminescence. In some embodiments, the first and the secondanalyte-binding agents are antigen-binding agents (e.g., antibodies). Insome embodiments, the first and the second antigen-binding agents bindto the same epitope of the analyte (e.g., a protein). In someembodiments, the first and the second antigen-binding agents bind toseparate epitopes of the analyte (e.g., a protein) that spatiallyoverlap. In some embodiments, the first and the second antigen-bindingagents bind to the separate epitopes of the analyte (e.g., a protein)that do not spatially overlap.

In certain embodiments, the disclosure provides a kit comprising aningestible device as described herein. In some embodiments, the kitfurther comprises instructions, e.g., for detecting or quantifying ananalyte in a sample.

In some embodiments, the disclosure provides methods for determining ananalyte in a sample. In certain embodiments, this disclosure provides amethod of detecting an analyte in a fluid sample of a subject,comprising: (1) providing an ingestible device; (2) transferring thefluid sample of the subject into the sampling chamber of the ingestibledevice in vivo; (3) irradiating the composition held in the samplingchamber of the ingestible device with light to excite thephotosensitizer; and (4) measuring total luminescence or rate of changeof luminescence emitted from the composition held in the samplingchamber of the ingestible device as a function of time, therebydetermining the level of the analyte in the fluid sample. In someembodiments, the method further comprises comparing the level of theanalyte in the fluid sample with the level of analyte in a referencesample (e.g., a reference sample obtained from a healthy subject). Insome embodiments, the level of the analyte in the sample is used todiagnose and/or monitor a disease or disorder in the subject.

In some embodiments, the disclosure provides a method of detecting ananalyte in a fluid sample of a subject, comprising: (1) providing aningestible device, the device comprising a sampling chamber that isconfigured to hold an absorbable material (e.g., an absorbable pad orsponge) having absorbed therein a composition, as described herein; (2)transferring the fluid sample of the subject into the sampling chamberof the ingestible device in vivo; (3) fully or partially saturating theabsorbable material held in the sampling chamber of the ingestibledevice with the fluid sample; (4) irradiating the absorbable materialheld in the sampling chamber of the ingestible device with light toexcite the photosensitizer; and (5) measuring total luminescence or rateof change of luminescence emitted from the composition held in thesampling chamber of the ingestible device as a function of time, therebydetermining the level of the analyte in the fluid sample. In someembodiments, the method further comprises comparing the level of theanalyte in the fluid sample with the level of analyte in a referencesample (e.g., a reference sample obtained from a healthy subject). Insome embodiments, the level of the analyte in the sample is used todiagnose and/or monitor a disease or disorder in the subject.

In some embodiments, the disclosure provides a method of assessing ormonitoring the need to treat a subject suffering from or at risk ofovergrowth of bacterial cells in the gastrointestinal (GI) tract,comprising: (1) providing an ingestible device for detecting an analyte;(2) transferring a fluid sample from the GI tract of the subject intothe sampling chamber of the ingestible device in vivo; (3) irradiatingthe composition held in the sampling chamber of the ingestible devicewith light to excite the photosensitizer; (4) measuring totalluminescence or rate of change of luminescence emitted from thecomposition held in the sampling chamber of the ingestible device as afunction of time; (5) correlating the total luminescence or the rate ofchange of luminescence as a function of time measured in step (4) to theamount of the analyte in the fluid sample; and (6) correlating theamount of the analyte in the fluid sample to the number of viablebacterial cells in the fluid sample. In some embodiments, a number ofviable bacterial cells determined in step (6) greater than a controlnumber of viable bacterial cells, indicates a need for treatment (e.g.,with an antibiotic agent described herein). In some embodiments, thecontrol number of viable bacterial cells is 10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, 10⁹, or more. For example, in some embodiments, a number of viablebacterial cells determined in step (6) greater that about 10³ CFU/mLindicates a need for treatment. In some embodiments, a number of viablebacterial cells determined in step (6) greater that about 10⁴ CFU/mLindicates a need for treatment. In some embodiments, a number of theviable bacterial cells determined in step (6) greater than about 10⁵CFU/mL indicates a need for treatment, e.g., with an antibiotic agent asdescribed herein. In some embodiments, a number of viable bacterialcells determined in step (6) greater that about 10⁶ or more CFU/mLindicates a need for treatment.

In some embodiments, the total luminescence or the rate of change ofluminescence as a function of time of the sponge is measured overmultiple time points for an extended period of time in step (4). Forinstance, in some embodiments, the total luminescence or rate of changeof luminescence as a function of time of the sample is measuredcontinuously for a period of 0-1800 minutes, 0-1600 minutes, 0-1500minutes, 0-1440 minutes, 0-1320 minutes, 0-1000 minutes, 0-900 minutes,0-800 minutes, 0-700 minutes, 0-600 minutes, 0-500 minutes, 0-400minutes, 0-350 minutes, 0-330 minutes, 0-300 minutes, 0-270 minutes, or0-220 minutes. In some embodiments, the total luminescence or the rateof change of luminescence as a function of time of said sample ismeasured continuously for a period of 0-330 minutes. In someembodiments, the method is performed in vivo. In some embodiments, themethod includes communicating the results of the onboard assay(s) to anex vivo receiver. In some embodiments, the total luminescence or therate of change of luminescence as a function of time of the sponge ismeasured over multiple time points for an extended period of time instep (5). For instance, in some embodiments, the total luminescence orrate of change of luminescence as a function of time of the sample ismeasured continuously for a period of 0-1800 minutes, 0-1600 minutes,0-1500 minutes, 0-1440 minutes, 0-1320 minutes, 0-1000 minutes, 0-900minutes, 0-800 minutes, 0-700 minutes, 0-600 minutes, 0-500 minutes,0-400 minutes, 0-350 minutes, 0-330 minutes, 0-300 minutes, 0-270minutes, or 0-220 minutes. In some embodiments, the total luminescenceor the rate of change of luminescence as a function of time of saidsample is measured continuously for a period of 0-330 minutes. In someembodiments, the method is performed in vivo. In some embodiments, themethod includes communicating the results of the onboard assay(s) to anex vivo receiver.

In some embodiments, the disclosure provides a method of assessing ormonitoring the need to treat a subject suffering from or at risk ofovergrowth of bacterial cells in the gastrointestinal tract, comprising:(1) providing an ingestible device for detecting an analyte, the devicecomprising a sampling chamber that is configured to hold an absorbablematerial (e.g., an absorbable pad or sponge) having absorbed therein acomposition, as described herein; (2) transferring a fluid sample fromthe GI tract of the subject into the sampling chamber of the ingestibledevice in vivo; (3) fully or partially saturating the absorbablematerial held in the sampling chamber of the ingestible device with thefluid sample; (4) irradiating the absorbable material held in thesampling chamber of the ingestible device with light to excite thephotosensitizer; (5) measuring total luminescence or rate of change ofluminescence emitted from the composition held in the sampling chamberof the ingestible device as a function of time; (6) correlating thetotal luminescence or the rate of change of luminescence as a functionof time measured in step (5) to the amount of the analyte in the fluidsample; and (7) correlating the amount of the analyte in the fluidsample to the number of viable bacterial cells in the fluid sample. Insome embodiments, a number of viable bacterial cells determined in step(7) greater than a control number of viable bacterial cells indicates aneed for treatment (e.g., with an antibiotic agent described herein). Insome embodiments, the control number of viable bacterial cells is 10³,10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or more. For example, in some embodiments,a number of viable bacterial cells determined in step (7) greater thatabout 10³ CFU/mL indicates a need for treatment. In some embodiments, anumber of viable bacterial cells determined in step (7) greater thatabout 10⁴ CFU/mL indicates a need for treatment. In some embodiments, anumber of the viable bacterial cells determined in step (7) greater thanabout 10⁵ CFU/mL indicates a need for treatment, e.g., with anantibiotic agent as described herein. In some embodiments, a number ofviable bacterial cells determined in step (7) greater that about 10⁶ ormore CFU/mL indicates a need for treatment.

In some embodiments, the disclosure, provides a method of measuring thepresence, absence or amount of one or more analytes from one or moresamples in the gastrointestinal tract. In some embodiments the one ormore analytes are measured multiple times, for example, at differenttime points or at different locations. In one embodiment, a singledevice measures one or more analytes or more time points or locations;thereby creating a “molecular map” of a physiological region.Measurements can be taken at any location in the gastrointestinal tract.For example, in one aspect, analytes from samples from one or more ofthe duodenum, jejunum, ileum, ascending colon, transverse colon ordescending colon can be measured to create a molecular map of the smalland large intestine. In one aspect, the sample is from the duodenum. Inone aspect, the sample is from the jejunum. In one aspect, the sample isfrom the ileum. In one aspect, the sample is from the ascending colon.In one aspect, the sample is from the transverse colon. In one aspect,the sample is from the descending colon.

In another aspect, a series of measurements can be taken over a shorterdistance of the gastrointestinal tract (e.g., the ileum) to create ahigher resolution molecular map. In some embodiments, previousendoscopic imaging may identify a diseased area for molecular mapping.For example, a gastroenterologist may use imaging (e.g., an endoscopeequipped with a camera) to identify the presence of Crohn's Disease inthe ileum and cecum of a patient, and the methods and techniques hereinmay be used to measure inflammation-associated analytes in this diseasedarea of the patient. In a related embodiment, theinflammation-associated analytes, or any analyte, may be measured everyone or more days to monitor disease flare-ups, or response totherapeutics.

Analytes

The compositions and methods described herein can be used to detect,analyze, and/or quantitate a variety of analytes in a human subject.“Analyte” as used herein refers to a compound or composition to bedetected in a sample. Exemplary analytes suitable for use herein includethose described in U.S. Pat. No. 6,251,581, which is incorporated byreference herein in its entirety. Broadly speaking, an analyte can beany substance (e.g., a substance with one or more antigens) capable ofbeing detected. An exemplary and non-limiting list of analytes includesligands, proteins, blood clotting factors, hormones, cytokines,polysaccharides, mucopolysaccharides, microorganisms (e.g., bacteria),microbial antigens, and therapeutic agents (including fragments andmetabolites thereof).

For instance, the analyte may be a ligand, which is monovalent(monoepitopic) or polyvalent (polyepitopic), usually antigenic orhaptenic, and is a single compound or plurality of compounds which shareat least one common epitopic or determinant site. The analyte can be apart of a cell such as bacteria or a cell bearing a blood group antigensuch as A, B, D, etc., a human leukocyte antigen (HLA), or other cellsurface antigen, or a microorganism, e.g., bacterium (e.g. a pathogenicbacterium), a fungus, protozoan, or a virus (e.g., a protein, a nucleicacid, a lipid, or a hormone). In some embodiments, the analyte can be apart of an exosome (e.g., a bacterial exosome). In some embodiments, theanalyte is derived from a subject (e.g., a human subject). In someembodiments, the analyte is derived from a microorganism present in thesubject. In some embodiments, the analyte is a nucleic acid (e.g., a DNAmolecule or a RNA molecule), a protein (e.g., a soluble protein, a cellsurface protein), or a fragment thereof, that can be detected using anyof the devices and methods provided herein.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,a polypeptide (i.e., protein) or a peptide, polysaccharides, nucleicacids (e.g., DNA or RNA), and combinations thereof. Such combinationsinclude components of bacteria, viruses, chromosomes, genes,mitochondria, nuclei, cell membranes, and the like.

In some embodiments, the polyepitopic ligand analytes have a molecularweight of at least about 5,000 Da, more usually at least about 10,000Da. In the poly(amino acid) category, the poly(amino acids) of interestmay generally have a molecular weight from about 5,000 Da to about5,000,000 Da, more usually from about 20,000 Da to 1,000,000 Da; amongthe hormones of interest, the molecular weights will usually range fromabout 5,000 Da to 60,000 Da.

In some embodiments, the monoepitopic ligand analytes generally have amolecular weight of from about 100 to 2,000 Da, more usually from 125 to1,000 Da.

A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc. Such proteins include,for example, immunoglobulins, cytokines, enzymes, hormones, cancerantigens, nutritional markers, tissue specific antigens, etc.

In some embodiments, the analyte is a protein. In some embodiments, theanalyte is a protein, e.g., an enzyme (e.g., a hemolysin, a protease, aphospholipase), a soluble protein, an exotoxin. In some embodiments, theanalyte is a fragment of a protein, a peptide, or an antigen. In someembodiments, the analyte is a peptide of at least 5 amino acids (e.g.,at least 6, at least 7, at least 8, at least 9, at least 10, at least25, at least, 50, or at least 100 amino acids). Exemplary lengthsinclude 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 50, 75, or 100 amino acids. Exemplaryclasses of protein analytes include, but are not limited to: protamines,histones, albumins, globulins, scleroproteins, phosphoproteins,mucoproteins, chromoproteins, lipoproteins, nucleoproteins,glycoproteins, T-cell receptors, proteoglycans, cell surface receptors,membrane-anchored proteins, transmembrane proteins, secreted proteins,HLA, and unclassified proteins.

In some embodiments, the analyte is an affimer (see, e.g., Tiede et al.(2017) eLife 6: e24903, which is expressly incorporated herein byreference).

Exemplary analytes include: Prealbumin, Albumin, α₁-Lipoprotein,α₁-Antitrypsin, α₁-Glycoprotein, Transcortin, 4.6S-Postalbumin,α₁-glycoprotein, α_(1x)-Glycoprotein, Thyroxin-binding globulin,Inter-α-trypsin-inhibitor, Gc-globulin (Gc 1-1, Gc 2-1, Gc 2-2),Haptoglobin (Hp 1-1, Hp 2-1, Hp 2-2), Ceruloplasmin, Cholinesterase,α₂-Lipoprotein(s), Myoglobin, C-Reactive Protein, α₂-Macroglobulin,α₂-HS-glycoprotein, Zn-α₂-glycoprotein, α₂-Neuramino-glycoprotein,Erythropoietin, β-lipoprotein, Transferrin, Hemopexin, Fibrinogen,Plasminogen, β₂-glycoprotein I, β₂-glycoprotein II, Immunoglobulin G(IgG) or γG-globulin, Immunoglobulin A (IgA) or γA-globulin,Immunoglobulin M (IgM) or γM-globulin, Immunoglobulin D (IgD) orγD-Globulin (γD), Immunoglobulin E (IgE) or γE-Globulin (γE), Free κ andλ, light chains, and Complement factors: C′1, (C′1q, C′1r, C′1s, C′2,C′3 (β₁A, α₂D), C′4, C'S, C′6, C′7, C′8, C′9.

Additional examples of analytes include tumor necrosis factor-α (TNFα),interleukin-12 (IL-12), IL-23, IL-6, α2β1 integrin, α1β1 integrin, α4β7integrin, integrin α4β1 (VLA-4), E-selectin, ICAM-1, α5β1 integrin, α4β1integrin, VLA-4, α2β1 integrin, α5β3 integrin, α5β5 integrin, αIIbβ3integrin, MAdCAM-1, SMAD7, JAK1, JAK2, JAK3, TYK-2, CHST15, IL-1, IL-1α,IL-1β, IL-18, IL-36α, IL-36β, IL-36γ, IL-38, IL-33, IL-13, CD40L, CD40,CD3γ, CD3δ, CD3ε, CD3ζ, TCR, TCRα, TCRβ, TCRδ, TCRγ, CD14, CD20, CD25,IL-2, IL-2 β chain, IL-2 γ chain, CD28, CD80, CD86, CD49, MMP1, CD89,IgA, CXCL10, CCL11, an ELR chemokine, CCR2, CCR9, CXCR3, CCR3, CCR5,CCL2, CCL8, CCL16, CCL25, CXCR1m CXCR2m CXCL1, CXCL2, CXCL3, CXCL4,CXCL5, CXCL6, CXCL7, and CXCL8, and a nucleic acid (e.g., mRNA) encodingany of the same.

In some embodiments, the analyte is a blood clotting factor. Exemplaryblood clotting factors include, but are not limited to:

International designation Name I Fibrinogen II Prothrombin IIa ThrombinIII Tissue thromboplastin V and VI Proaccelerin, accelerator globulinVII Proconvertin VIII Antihemophilic globulin (AHG) IX Christmas factorplasma thromboplastin component (PTC) X Stuart-Prower factor,autoprothrombin III XI Plasma thromboplastin antecedent (PTA) XIIHagemane factor XIII Fibrin-stabilizing factor

In some embodiments, the analyte is a hormone. Exemplary hormonesinclude, but are not limited to: Peptide and Protein Hormones,Parathyroid hormone, (parathromone), Thyrocalcitonin, Insulin, Glucagon,Relaxin, Erythropoietin, Melanotropin (melancyte-stimulating hormone;intermedin), Somatotropin (growth hormone), Corticotropin(adrenocorticotropic hormone), Thyrotropin, Follicle-stimulatinghormone, Luteinizing hormone (interstitial cell-stimulating hormone),Luteomammotropic hormone (luteotropin, prolactin), Gonadotropin(chorionic gonadotropin), Secretin, Gastrin, Angiotensin I and II,Bradykinin, and Human placental lactogen, thyroxine, cortisol,triiodothyronine, testosterone, estradiol, estrone, progestrone,luteinizing hormone-releasing hormone (LHRH), and immunosuppressantssuch as cyclosporin, FK506, mycophenolic acid, and so forth.

In some embodiments, the analyte is a peptide hormone (e.g., a peptidehormone from the neurohypophysis). Exemplary peptide hormones from theneurohypophysis include, but are not limited to: Oxytocin, Vasopressin,and releasing factors (RF) (e.g., corticotropin releasing factor (CRF),luteinizing hormone releasing factor (LRF), thyrotropin releasing factor(TRF), Somatotropin-RF, growth hormone releasing factor (GRF), folliclestimulating hormone-releasing factor (FSH-RF), prolactin inhibitingfactor (PIF), and melanocyte stimulating hormone inhibiting factor(MIF)).

In some embodiments, the analyte is a cytokine or a chemokine. Exemplarycytokines include, but are not limited to: interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-6 (IL-6), epidermal growth factor(EGF), tumor necrosis factor (TNF, e.g., TNF-α or TNF-β), and nervegrowth factor (NGF).

In some embodiments, the analyte is a cancer antigen. Exemplary cancerantigens include, but are not limited to: prostate-specific antigen(PSA), carcinoembryonic antigen (CEA), α-fetoprotein, Acid phosphatase,CA19.9, and CA125.

In some embodiments, the analyte is a tissue-specific antigen. Exemplarytissue specific antigens include, but are not limited to: alkalinephosphatase, myoglobin, CPK-MB, calcitonin, and myelin basic protein.

In some embodiments, the analyte is a mucopolysaccharide or apolysaccharide.

In some embodiments, the analyte is a microorganism, or a moleculederived from or produced by a microorganism (e.g., a bacteria, a virus,prion, or a protozoan). For example, in some embodiments, the analyte isa molecule (e.g., a protein or a nucleic acid) that is specific for aparticular microbial genus, species, or strain (e.g., a specificbacterial genus, species, or strain). In some embodiments, themicroorganism is pathogenic (i.e., causes disease). In some embodiments,the microorganism is non-pathogenic (e.g., a commensal microorganism).Exemplary microorganisms include, but are not limited to:

Corynebacteria Corynebacterium diphtheria Pneumococci Diplococcuspneumoniae Streptococci Streptococcus pyrogenes Streptococcus salivarusStaphylococci Staphylococcus aureus Staphylococcus albus NeisseriaNeisseria meningitidis Neisseria gonorrhea EnterobacteriaciaeEscherichia coli Aerobacter aerogenes The coliform Klebsiella pneumoniaebacteria Salmonella typhosa Salmonella choleraesuis The SalmonellaeSalmonella typhimurium Shigella dysenteria Shigella schmitzii Shigellaarabinotarda The Shigellae Shigella flexneri Shigella boydii Shigellasonnei Other enteric bacilli Proteus vulgaris Proteus mirabilis Proteusspecies Proteus morgani Pseudomonas aeruginosa Alcaligenes faecalisVibrio cholerae Hemophilus-Bordetella group Rhizopus oryzae Hemophilusinfluenza, H. ducryi Rhizopus arrhizua Phycomycetes Hemophilushemophilus Rhizopus nigricans Hemophilus aegypticus Sporotrichumschenkii Hemophilus parainfluenza Flonsecaea pedrosoi Bordetellapertussis Fonsecacea compact Pasteurellae Fonsecacea dermatidisPasteurella pestis Cladosporium carrionii Pasteurella tulareusisPhialophora verrucosa Brucellae Aspergillus nidulans Brucella melltensisMadurella mycetomi Brucella abortus Madurella grisea Brucella suisAllescheria boydii Aerobic Spore-forming Bacilli Phialophora jeanselmeiBacillus anthracis Microsporum gypseum Bacillus subtilis Trichophytonmentagrophytes Bacillus megaterium Keratinomyces ajelloi Bacillus cereusMicrosporum canis Anaerobic Spore-forming Bacilli Trichophyton rubrumClostridium botulinum Microsporum adouini Clostridium tetani VirusesClostridium perfringens Adenoviruses Clostridium novyi Herpes VirusesClostridium septicum Herpes simplex Clostridium histoyticum Varicella(Chicken pox) Clostridium tertium Herpes Zoster (Shingles) Clostridiumbifermentans Virus B Clostridium sporogenes Cytomegalovirus MycobacteriaPox Viruses Mycobacterium tuberculosis hominis Variola (smallpox)Mycobacterium bovis Vaccinia Mycobacterium avium Poxvirus bovisMycobacterium leprae Paravaccinia Mycobacterium paratuberculosisMolluscum contagiosum Actinomycetes (fungus-ike bacteria) PicornavirusesActinomyces Isaeli Poliovirus Actinomyces bovis CoxsackievirusActinomyces naeslundii Echoviruses Nocardia asteroides RhinovirusesNocardia brasiliensis Myxoviruses The Spirochetes Influenza(A, B, and C)Treponema pallidum Parainfluenza (1-4) Treponema pertenue Mumps VirusSpirillum minus Streptobacillus monoiliformis Newcastle Disease VirusTreponema carateum Measles Virus Borrelia recurrentis Rinderpest VirusLeptospira icterohemorrhagiae Canine Distemper Virus Leptospira canicolaRespiratory Syncytial Virus Trypanasomes Rubella Virus MycoplasmasArboviruses Mycoplasma pneumoniae Other pathogens Eastern EquineEncephalitis Virus Listeria monocytogenes Western Equine EncephalitisVirus Erysipeothrix rhusiopathiae Sindbis Virus Streptobacillusmoniliformis Chikugunya Virus Donvania granulomatis Semliki Forest VirusEntamoeba histolytica Mayora Virus Plasmodium falciparum St. LouisEncephalitis Plasmodium japonicum California Encephalitis VirusBartonella bacilliformis Colorado Tick Fever Virus Rickettsia(bacteria-like parasites) Yellow Fever Virus Rickettsia prowazekiiDengue Virus Rickettsia mooseri Reoviruses Rickettsia rickettsiiReovirus Types 1-3 Rickettsia conori Retroviruses Rickettsia australisHuman Immunodeficiency Rickettsia sibiricus Viruses I and II (HTLV)Rickettsia akari Human T-cell Lymphotrophic Rickettsia tsutsugamushiVirus I & II (HIV) Rickettsia burnetti Hepatitis Rickettsia quintanaHepatitis A Virus Chlamydia (unclassifiable parasites Hepatitis B Virusbacterial/viral) Hepatitis C Virus Chlamydia agents (naming uncertain)Tumor Viruses Chlamydia trachomatis Fungi Rauscher Leukemia VirusCryptococcus neoformans Gross Virus Blastomyces dermatidis MaloneyLeukemia Virus Histoplasma capsulatum Coccidioides immitis HumanPapilloma Virus Paracoccidioides brasliensis Candida albicansAspergillus fumigatus Mucor corymbifer (Absidia corymbifera)

In some embodiments, the analyte is a bacterium. Exemplary bacteriainclude, but are not limited to: Escherichia coli (or E. coli), Bacillusanthracis, Bacillus cereus, Clostridium botulinum, Clostridiumdifficile, Yersinia pestis, Yersinia enterocolitica, Francisellatularensis, Brucella species, Clostridium perfringens, Burkholderiamallei, Burkholderia pseudomallei, Staphylococcus species, Mycobacteriumspecies, Group A Streptococcus, Group B Streptococcus, Streptococcuspneumoniae, Helicobacter pylori, Salmonella enteritidis, Mycoplasmahominis, Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans,Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium leprae, Rickettsia rickettsii,Rickettsia akari, Rickettsia prowazekii, Rickettsia canada, Bacillussubtilis, Bacillus subtilis niger, Bacillus thuringiensis, Coxiellaburnetti, Faecalibacterium prausnitzii (also known as Bacteroidespraussnitzii), Roseburia hominis, Eubacterium rectale, Dialisterinvisus, Ruminococcus albus, Ruminococcus callidus, and Ruminococcusbromii. Additional exemplary bacteria include bacteria of the phylaFirmicutes (e.g., Clostridium clusters XIVa and IV), bacteria of thephyla Bacteroidetes (e.g., Bacteroides fragilis or Bacteroidesvulgatus), and bacteria of the phyla Actinobacteria (e.g.,Coriobacteriaceae spp. or Bifidobacterium adolescentis). Bacteria of theClostridium cluster XIVa includes species belonging to, for example, theClostridium, Ruminococcus, Lachnospira, Roseburia, Eubacterium,Coprococcus, Dorea, and Butyrivibrio genera. Bacteria of the Clostridiumcluster IV includes species belonging to, for example, the Clostridium,Ruminococcus, Eubacterium and Anaerofilum genera. In some embodiments,the analyte is Candida, e.g., Candida albicans. In some embodiments, theanalyte is a byproduct from a bacterium or other microorganism, e.g.,helminth ova, enterotoxin (Clostridium difficile toxin A; TcdA) orcytotoxin (Clostridium difficile toxin B; TcdB).

In some embodiments, the bacterium is a pathogenic bacterium.Non-limiting examples of pathogenic bacteria belong to the generaBacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia,Chlamydophila, Clostridium, Corynebacterium, Enterobacter, Enterococcus,Escherichia, Francisella, Haemophilus, Helicobacter, Legionella,Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,Treponema, Vibrio, and Yersinia. Non-limiting examples of specificpathogenic bacterial species include a strain of Bacillus anthracis, astrain of a strain of Bordetella pertussis, a strain of a strain ofBorrelia burgdorferi, a strain of a strain of Brucella abortus, a strainof a strain of Brucella canis, a strain of a strain of Brucellamelitensis, a strain of a strain of Brucella suis, a strain of a strainof Campylobacter jejuni, a strain of Chlamydia pneumoniae, a strain ofChlamydia trachomatis, a strain of Chlamydophila psittaci, a strain ofClostridium botulinum, a strain of Clostridium difficile, a strain ofClostridium perfringens, a strain of Clostridium tetani, a strain ofCorynebacterium diphtheria, a strain of Enterobacter sakazakii, a strainof Enterococcus faecalis, a strain of Enterococcus faecium, a strain ofEscherichia coli (e.g., E. coli O157 H7), a strain of Francisellatularensis, a strain of Haemophilus influenza, a strain of Helicobacterpylori, a strain of Legionella pneumophila, a strain of Leptospirainterrogans, a strain of Listeria monocytogenes, a strain ofMycobacterium leprae, a strain of Mycobacterium tuberculosis, a strainof Mycobacterium ulcerans, a strain of Mycoplasma pneumonia, a strain ofNeisseria gonorrhoeae, a strain of Neisseria meningitides, a strain ofPseudomonas aeruginosa, a strain of Rickettsia rickettsia, a strain ofSalmonella typhi and Salmonella typhimurium, a strain of Shigellasonnei, a strain of Staphylococcus aureus, a strain of Staphylococcusepidermidis, a strain of Staphylococcus saprophyticus, a strain ofStreptococcus agalactiae, a strain of Streptococcus pneumonia, a strainof Streptococcus pyogenes, a strain of Treponema pallidum, a strain ofVibrio cholera, a strain of Yersinia enterocolitica, and, a strain ofYersinia pestis.

In some embodiments, the bacterium is a commensal bacterium (e.g., aprobiotic). In some embodiments, the bacterium has been previouslyadministered to a subject, e.g., as a live biotherapeutic agent.Exemplary commensal bacteria include, but are not limited to,Faecalibacterium prausnitzii (also referred to as Bacteroidespraussnitzii), Roseburia hominis, Eubacterium rectale, Dialisterinvisus, Ruminococcus albus, Ruminococcus gnavus, Ruminococcus torques,Ruminococcus callidus, and Ruminococcus bromii.

In some embodiments, the analyte is a virus. In some embodiments, thevirus is a pathogenic virus. Non-limiting examples of pathogenic virusesbelong to the families Adenoviridae, Picornaviridae, Herpesviridae,Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae,Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, andTogaviridae.

In some embodiments, the analyte is a fungus. In some embodiments, thefungi is a pathogenic fungus. Non-limiting examples of pathogenic fungibelong to the genera Asperfillus, Canidia, Cryptococcus, Histoplasma,Pneumocystis, and Stachybotrys. Non-limiting examples of specificpathogenic fungi species include a strain of Aspergillus clavatus,Aspergillus fumigatus, Aspergillus flavus, Canidia albicans,Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii,Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis jirovecii,Pneumocystis carinii, and Stachybotrys chartarum.

In some embodiments, the analyte is a protozoan. In some embodiments,the analyte is a pathogenic protozoan. Non-limiting examples ofpathogenic protozoa belong to the genera Acanthamoeba, Balamuthia,Cryptosporidium, Dientamoeba, Endolimax, Entamoeba, Giardia, Iodamoeba,Leishmania, Naegleria, Plasmodium, Sappinia, Toxoplasma, Trichomonas,and Trypanosoma. Non-limiting examples of specific pathogenic protozoaspecies include a strain of Acanthamoeba spp., Balamuthia mandrillaris,Cryptosporidium canis, Cryptosporidium fells, Cryptosporidium hominis,Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidiumparvum, Dientamoeba fragilis, Endolimax nana, Entamoeba dispar,Entamoeba hartmanni, Entamoeba histolytica, Entamoeba coli, Entamoebamoshkovskii, Giardia lamblia, Iodamoeba butschlii, Leishmaniaaethiopica, Leishmania braziliensis, Leishmania chagasi, Leishmaniadonovani, Leishmania infantum, Leishmania major, Leishmania mexicana,Leishmania tropica, Naegleria fowleri, Plasmodium falciparum, Plasmodiumknowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax,Sappinia diploidea, Toxoplasma gondii, Trichomonas vaginalis,Trypanosoma brucei, and Trypanosoma cruzi.

In some embodiments, the analyte is secreted by or expressed on the cellsurface of a microorganism (e.g., a bacterium, a colonic bacterium, aviable bacterium, a dead bacterium, a parasite (e.g., Giardia lamblia,Cryptosporidium, Cystoisosporiasis belli, and Balantidium coli), a virus(e.g., a herpes virus, a cytomegalovirus, a herpes simplex virus, anEpstein-Barr virus, a human papilloma virus, a rotavirus, a humanherpesvirus-8; Goodgame (1999) Curr. Gastroenterol. Rep. 1(4): 292-300).In some embodiments, the analyte is secreted by or expressed on the cellsurface of a Gram-negative bacterium (e.g., E. coli, Helicobacterpylori). In some embodiments, the analyte is secreted by or expressed onthe cell surface (e.g., a bacterial surface epitope) of a Gram-positivebacterium (e.g., Staphylococcus aureus, Clostridium botulinum,Clostridium difficile).

In some embodiments, the analyte is a molecule expressed on the surfaceof a bacterial cell (e.g., a bacterial cell surface protein). In someembodiments, the analyte is a bacterial toxin (e.g., TcdA and/or TcdBfrom Clostridium difficile). In some embodiments, the analyte is CFA/Ifimbriae, flagella, lipopolysaccharide (LPS), lipoteichoic acid, or apeptidoglycan. Non-limiting examples of bacterium that may express ananalyte that can be detected using any of the devices and methodsdescribed herein include: Bacillus anthraces, Bacillus cereus,Clostridium botulinum, Clostridium difficile, Escherichia coli, Yersiniapestis, Yersinia enterocolitica, Francisella tularensis, Brucellaspecies, Clostridium perfringens, Burkholderia mallei, Burkholderiapseudomallei, Helicobacter pylori, Staphylococcus species, Mycobacteriumspecies, Group A Streptococcus, Group B Streptococcus, Streptococcuspneumoniae, Francisella tularensis, Salmonella enteritidis, Mycoplasmahominis, Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans,Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium leprae, Rickettsia rickettsia,Rickettsia akari, Rickettsia prowazekii, Rickettsia canada, Bacillussubtilis, Bacillus subtilis niger, Bacillus thuringiensis, Coxiellabumetti, Candida albicans, Bacteroides fragilis, Leptospira interrogans,Listeria monocytogenes, Pasteurella multocida, Salmonella typhi,Salmonella typhimurium, Shigella dysenteriae, Shigella flexneria,Shigella sonnei, Vibrio cholera, and Vibrio parahaemolyticus.

In some embodiments, the analyte is a byproduct from a bacterium oranother microorganism, e.g., helminth ova, enterotoxin (Clostridiumdifficile toxin A; TcdA), cytotoxin (Clostridium difficile toxin B;TcdB), ammonia. In some embodiments, the analyte is an antigen from amicroorganism (e.g., a bacteria, virus, prion, fungus, protozoan or aparasite).

In some embodiments, the analytes include drugs, metabolites,pesticides, pollutants, and the like. Included among drugs of interestare the alkaloids. Among the alkaloids are morphine alkaloids, whichincludes morphine, codeine, heroin, dextromethorphan, their derivativesand metabolites; cocaine alkaloids, which include cocaine and benzylecgonine, their derivatives and metabolites; ergot alkaloids, whichinclude the diethylamide of lysergic acid; steroid alkaloids; iminazoylalkaloids; quinazoline alkaloids; isoquinoline alkaloids; quinolinealkaloids, which include quinine and quinidine; diterpene alkaloids,their derivatives and metabolites.

In some embodiments, the analyte is a steroid selected from theestrogens, androgens, andreocortical steroids, bile acids, cardiotonicglycosides and aglycones, which includes digoxin and digoxigenin,saponins and sapogenins, their derivatives and metabolites. Alsoincluded are the steroid mimetic substances, such as diethylstilbestrol.

In some embodiments, the analyte is a bile acid. In some embodiments,the presence, absence, and/or a specific level of one or more bile acidsin the GI tract of a subject is indicative of a condition or diseasestate (e.g., a GI disorder and/or a non-GI disorder (e.g., a systemicdisorder). For example, in some embodiments, the compositions andmethods described herein may be used to detect and/or quantify a bileacid in the GI tract of the subject to diagnose a condition such as bileacid malabsorption (also known as bile acid diarrhea). In someembodiments, the analyte is a metabolite in the serotonin, tryptophanand/or kynurenine pathways, including but not limited to, serotonin(5-HT), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan(5-HTP), kynurenine (K), kynurenic acid (KA), 3-hydroxykynurenine(3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid, anthranilicacid, and combinations thereof 5-HT is a molecule that plays a role inthe regulation of gastrointestinal motility, secretion, and sensation.Imbalances in the levels of 5-HT are associated with several diseasesincluding inflammatory bowel syndrome (IBS), autism, gastric ulcerformation, non-cardiac chest pain, and functional dyspepsia (see, e.g.,Faure et al. (2010) Gastroenterology 139(1): 249-58 and Muller et al.(2016) Neuroscience 321: 24-41, and International Publication No. WO2014/188377, each of which are incorporated herein by reference).Conversion of metabolites within the serotonin, tryptophan and/orkynurenine pathways affects the levels of 5-HT in a subject. Therefore,measuring the levels of one or more of the metabolites in this pathwaymay be used for the diagnosis, management and treatment of a disease ordisorder associated with 5-HT imbalance including but not limited toIBS, autism, carcinoid syndrome, depression, hypertension, Alzheimer'sdisease, constipation, migraine, and serotonin syndrome. One or moreanalytes in the serotonin, tryptophan and/or kynurenine pathways can bedetected and/or quantitated using, for example, methods andanalyte-binding agents that bind to these metabolites including, e.g.,antibodies, known in the art (see, e.g., International Publication No.WO2014/188377, the entire contents of which are expressly incorporatedherein by reference).

In some embodiments, the analyte is a lactam having from 5 to 6 annularmembers selected from barbituates, e.g., phenobarbital and secobarbital,diphenylhydantonin, primidone, ethosuximide, and metabolites thereof.

In some embodiments, the analyte is an aminoalkylbenzene, with alkyl offrom 2 to 3 carbon atoms, selected from the amphetamines;catecholamines, which includes ephedrine, L-dopa, epinephrine; narceine;papaverine; and metabolites thereof.

In some embodiments, the analyte is a benzheterocyclic selected fromoxazepam, chlorpromazine, tegretol, their derivatives and metabolites,the heterocyclic rings being azepines, diazepines and phenothiazines.

In some embodiments, the analyte is a purine selected from theophylline,caffeine, their metabolites and derivatives.

In some embodiments, the analyte is marijuana, cannabinol ortetrahydrocannabinol.

In some embodiments, the analyte is a vitamin such as vitamin A, vitaminB, e.g. vitamin B12, vitamin C, vitamin D, vitamin E and vitamin K,folic acid, thiamine.

In some embodiments, the analyte is selected from prostaglandins, whichdiffer by the degree and sites of hydroxylation and unsaturation.

In some embodiments, the analyte is a tricyclic antidepressant selectedfrom imipramine, dismethylimipramine, amitriptyline, nortriptyline,protriptyline, trimipramine, chlomipramine, doxepine, anddesmethyldoxepin.

In some embodiments, the analyte is selected from anti-neoplastics,including methotrexate.

In some embodiments, the analyte is an antibiotic as described herein,including, but not limited to, penicillin, chloromycetin, actinomycetin,tetracycline, terramycin, and metabolites and derivatives.

In some embodiments, the analyte is a nucleoside and nucleotide selectedfrom ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidine withtheir appropriate sugar and phosphate substituents.

In some embodiments, the analyte is selected from methadone,meprobamate, serotonin, meperidine, lidocaine, procainamide,acetylprocainamide, propranolol, griseofulvin, valproic acid,butyrophenones, antihistamines, chloramphenicol, anticholinergic drugs,such as atropine, their metabolites and derivatives.

In some embodiments, the analyte is a metabolite related to a diseasedstate. Such metabolites include, but are not limited to spermine,galactose, phenylpyruvic acid, and porphyrin Type 1.

In some embodiments, the analyte is an aminoglycoside, such asgentamicin, kanamicin, tobramycin, or amikacin.

In some embodiments, the analyte is a pesticide. Among pesticides ofinterest are polyhalogenated biphenyls, phosphate esters,thiophosphates, carbamates, polyhalogenated sulfenamides, theirmetabolites and derivatives.

In some embodiments, the analyte has a molecular weight of about 500 Dato about 1,000,000 Da (e.g., about 500 to about 500,000 Da, about 1,000to about 100,000 Da).

In some embodiments, the analyte is a receptor, with a molecular weightranging from 10,000 to 2×10⁸ Da, more usually from 10,000 to 10⁶ Da. Forimmunoglobulins, IgA, IgG, IgE and IgM, the molecular weights willgenerally vary from about 160,000 Da to about 10⁶ Da. Enzymes willnormally range in molecular weight from about 10,000 Da to about1,000,000 Da. Natural receptors vary widely, generally having amolecular weight of at least about 25,000 Da and may be 10⁶ or higherDa, including such materials as avidin, DNA, RNA, thyroxine bindingglobulin, thyroxine binding prealbumin, transcortin, etc.

In some embodiments, the term “analyte” further includes polynucleotideanalytes such as those polynucleotides defined below. These includem-RNA, r-RNA, t-RNA, DNA, DNA-RNA duplexes, etc. The term analyte alsoincludes polynucleotide-binding agents, such as, for example,restriction enzymes, trascription factors, transcription activators,transcription repressors, nucleases, polymerases, histones, DNA repairenzymes, intercalating gagents, chemotherapeutic agents, and the like.

In some embodiments, the analyte may be a molecule found directly in asample such as a body fluid from a host. The sample can be examineddirectly or may be pretreated to render the analyte more readilydetectable. Furthermore, the analyte of interest may be determined bydetecting an agent probative of the analyte of interest (i.e., ananalyte-binding agent), such as a specific binding pair membercomplementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay.

In some embodiments, the analyte a nucleic acid (e.g., a bacterial DNAmolecule or a bacterial RNA molecule (e.g., a bacterial tRNA, atransfer-messenger RNA (tmRNA)). See, e.g., Sjostrom et al. (2015)Scientific Reports 5: 15329; Ghosal (2017) Microbial Pathogenesis 104:161-163; Shen et al. (2012) Cell Host Microbe. 12(4): 509-520.

In some embodiments, the analyte is a component of an outer membranevesicle (OMV) (e.g., an OmpU protein, Elluri et al. (2014) PloS One 9:e106731). See, e.g., Kulp and Kuehn (2010) Annual Review of microbiology64: 163-184; Berleman and Auer (2013) Environmental microbiology 15:347-354; Wai et al. (1995) Microbiology and immunology 39: 451-456;Lindmark et al. (2009) BMC microbiology 9: 220; Sjostrom et al. (2015)Scientific Reports 5: 15329.

In some embodiments, the analyte is G-CSF, which can stimulate the bonemarrow to produce granulocytes and stem cells and release them into thebloodstream.

In some embodiments, the analyte is an enzyme such as glutathioneS-transferase. For example, the ingestible device can include P28GST, a28 kDa helminth protein from Schistosoma with potent immunogenic andantioxidant properties. P28GST prevents intestinal inflammation inexperimental colitis through a Th2-type response with mucosaleosinophils and can be recombinantly produced (e.g., in S. cerevisiae).See, for example, U.S. Pat. No. 9,593,313, Driss et al., MucosalImmunology, 2016 9, 322-335; and Capron et al., Gastroenterology,146(5):S-638.

In some embodiments, the analyte is a metabolite in the serotonin,tryptophan and/or kynurenine pathways, including but not limited to,serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA),5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenic acid (KA),3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA),quinolinic acid, anthranilic acid, and combinations thereof.

In some embodiments, analytes are therapeutic agents or drugs. In someembodiments, analytes are biomarkers. The therapeutic agents disclosedherein are can also be analytes. Examples of biomarkers are providedherein.

In some embodiments, analytes are therapeutic agents, fragments thereof,and metabolites thereof (e.g., antibiotics). In some embodiments, theanalytes are antibodies. In some embodiments, the analytes areantibiotics. Additional exemplary analytes (e.g., antibodies andantibiotics) are provided below.

a. Antibodies

In some embodiments, the analyte or the analyte-binding agent is anantibody. An “antibody” is an immunoglobulin molecule capable ofspecific binding to a target, such as a carbohydrate, polynucleotide,lipid, polypeptide, etc., through at least one antigen recognition site,located in the variable region of the immunoglobulin molecule. As usedherein, the term encompasses not only intact polyclonal or monoclonalantibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv),single chain (ScFv) and domain antibodies), and fusion proteinsincluding an antibody portion, and any other modified configuration ofthe immunoglobulin molecule that includes an antigen recognition site.The term antibody includes antibody fragments (e.g., antigen-bindingfragments) such as an Fv fragment, a Fab fragment, a F(ab′)2 fragment,and a Fab′ fragment. Additional examples of antigen-binding fragmentsinclude an antigen-binding fragment of an IgG (e.g., an antigen-bindingfragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-bindingfragment of a human or humanized IgG, e.g., human or humanized IgG1,IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., anantigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-bindingfragment of a human or humanized IgA, e.g., a human or humanized IgA1 orIgA2); an antigen-binding fragment of an IgD (e.g., an antigen-bindingfragment of a human or humanized IgD); an antigen-binding fragment of anIgE (e.g., an antigen-binding fragment of a human or humanized IgE); oran antigen-binding fragment of an IgM (e.g., an antigen-binding fragmentof a human or humanized IgM). An antibody includes an antibody of anyclass, such as IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class. Depending on the antibody aminoacid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies including the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, 1975, Nature 256:495, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature 348:552-554, for example.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FR) connected by three complementarity determining regions(CDRs) that contain hypervariable regions. The CDRs in each chain areheld together in close proximity by the FRs and, with the CDRs from theother chain, contribute to the formation of the antigen-binding site ofantibodies. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-Lazikaniet al, 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR mayrefer to CDRs defined by either approach or by a combination of bothapproaches.

As known in the art, a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

A “derivative” refers to any polypeptide (e.g., an antibody) having asubstantially identical amino acid sequence to the naturally occurringpolypeptide, in which one or more amino acids have been modified at sidegroups of the amino acids (e.g., a biotinylated protein or antibody).The term “derivative” shall also include any polypeptide (e.g., anantibody) which has one or more amino acids deleted from, added to, orsubstituted from the natural polypeptide sequence, but which retains asubstantial amino acid sequence homology to the natural sequence. Asubstantial sequence homology is any homology greater than 50 percent.

In some embodiments, the antibody can be a humanized antibody, achimeric antibody, a multivalent antibody, or a fragment thereof. Insome embodiments, an antibody can be a scFv-Fc (Sokolowska-Wedzina etal., Mol. Cancer Res. 15(8):1040-1050, 2017), a VHH domain (Li et al.,Immunol. Lett. 188:89-95, 2017), a VNAR domain (Hasler et al., Mol.Immunol. 75:28-37, 2016), a (scFv)₂, a minibody (Kim et al., PLoS One10(1):e113442, 2014), or a BiTE. In some embodiments, an antibody can bea DVD-Ig (Wu et al., Nat. Biotechnol. 25(11):1290-1297, 2007; WO08/024188; WO 07/024715), and a dual-affinity re-targeting antibody(DART) (Tsai et al., Mol. Ther. Oncolytics 3:15024, 2016), a triomab(Chelius et al., MAbs 2(3):309-319, 2010), kih IgG with a common LC(Kontermann et al., Drug Discovery Today 20(7):838-847, 2015), acrossmab (Regula et al., EMBO Mol. Med. 9(7):985, 2017), an ortho-FabIgG (Kontermann et al., Drug Discovery Today 20(7):838-847, 2015), a2-in-1-IgG (Kontermann et al., Drug Discovery Today 20(7):838-847,2015), IgG-scFv (Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014),scFv2-Fc (Natsume et al., J. Biochem. 140(3):359-368, 2006), abi-nanobody (Kontermann et al., Drug Discovery Today 20(7):838-847,2015), tanden antibody (Kontermann et al., Drug Discovery Today20(7):838-847, 2015), a DART-Fc (Kontermann et al., Drug Discovery Today20(7):838-847, 2015), a scFv-HSA-scFv (Kontermann et al., Drug DiscoveryToday 20(7):838-847, 2015), DNL-Fab3 (Kontermann et al., Drug DiscoveryToday 20(7):838-847, 2015), DAF (two-in-one or four-in-one), DutaMab,DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pairantibody, Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, Fcab,kλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv,scFv-(L)-IgG, IgG (L,H)-Fc, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIHIgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody(e.g., antibodies derived from Camelus bactriamus, Calelus dromaderius,or Lama paccos) (U.S. Pat. No. 5,759,808; Stijlemans et al., J. Biol.Chem. 279:1256-1261, 2004; Dumoulin et al., Nature 424:783-788, 2003;and Pleschberger et al., Bioconjugate Chem. 14:440-448, 2003),nanobody-HSA, a diabody (e.g., Poljak, Structure 2(12):1121-1123, 1994;Hudson et al., J. Immunol. Methods 23(1-2):177-189, 1999), a TandAb(Reusch et al., mAbs 6(3):727-738, 2014), scDiabody (Cuesta et al.,Trends in Biotechnol. 28(7):355-362, 2010), scDiabody-CH3 (Sanz et al.,Trends in Immunol. 25(2):85-91, 2004), Diabody-CH3 (Guo et al.), TripleBody, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv,scFv-CH-CL-scFv, F(ab′)2-scFV2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb,scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody (Huston et al.,Human Antibodies 10(3-4):127-142, 2001; Wheeler et al., Mol. Ther.8(3):355-366, 2003; Stocks, Drug Discov. Today 9(22):960-966, 2004),dock and lock bispecific antibody, ImmTAC, HSAbody, scDiabody-HSA,tandem scFv, IgG-IgG, Cov-X-Body, and scFv1-PEG-scFv2.

In some embodiments, an antibody can be an IgNAR, a bispecific antibody(Milstein and Cuello, Nature 305:537-539, 1983; Suresh et al., Methodsin Enzymology 121:210, 1986; WO 96/27011; Brennan et al., Science229:81, 1985; Shalaby et al., J. Exp. Med. 175:217-225, 1992; Kolstelnyet al., J. Immunol. 148(5):1547-1553, 1992; Hollinger et al., Proc.Natl. Acad. Sci. U.S.A. 90:6444-6448, 1993; Gruber et al., J. Immunol.152:5368, 1994; Tutt et al., J. Immunol. 147:60, 1991), a bispecificdiabody, a triabody (Schoonooghe et al., BMC Biotechnol. 9:70, 2009), atetrabody, scFv-Fc knobs-into-holes, a scFv-Fc-scFv, a (Fab′scFv)₂, aV-IgG, a IvG-V, a dual V domain IgG, a heavy chain immunoglobulin or acamelid (Holt et al., Trends Biotechnol. 21(11):484-490, 2003), anintrabody, a monoclonal antibody (e.g., a human or humanized monoclonalantibody), a heteroconjugate antibody (e.g., U.S. Pat. No. 4,676,980), alinear antibody (Zapata et al., Protein Eng. 8(10:1057-1062, 1995), atrispecific antibody (Tutt et al., J. Immunol. 147:60, 1991), aFabs-in-Tandem immunoglobulin (WO 15/103072), or a humanized camelidantibody.

In some embodiments, the antibody binds specifically to a metabolite inthe serotonin, tryptophan and/or kynurenine pathways, including but notlimited to, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA),5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenic acid (KA),3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA),quinolinic acid, anthranilic acid. Exemplary antibodies that bind tometabolites in these pathways are disclosed, for example, inInternational Publication No. WO2014/188377, the entire contents ofwhich are incorporated herein by reference.

In some embodiments, the antibody is specific for a particular genus,species, or strain of a microorganism, and may therefore be used for thedetection, analysis and/or quantitation of the microorganism using thedetection methods described below. In some embodiments, the antibodyspecifically binds to a surface-specific biomolecule (e.g., a pilussubunit or a flagella protein) present in a particular genus, species orstrain of microorganism, and does not cross-react with othermicroorganisms. In some embodiments, these antibodies may be used in themethods described herein to diagnose a subject with a particularinfection or disease, or to monitor an infection (e.g., during or aftertreatment). In some embodiments, the antibody specifically binds to anantigen present in a particular genera, species or strain of amicroorganism. Exemplary antigens, the corresponding microorganism thatcan be detected, and the disease caused by the microorganism (inparentheticals) include: outer membrane protein A OmpA (Acinetobacterbaumannii, Acinetobacter infections)); HIV p24 antigen, HIV Eenvelopeproteins (Gp120, Gp41, Gp160) (HIV (Human immunodeficiency virus), AIDS(Acquired immunodeficiency syndrome)); galactose-inhibitable adherenceprotein GIAP, 29 kDa antigen Eh29, GaVGaINAc lectin, protein CRT, 125kDa immunodominant antigen, protein M17, adhesin ADH112, protein STIRP(Entamoeba histolytica, Amoebiasis); protective Antigen PA, edema factorEF, lethal facotor LF, the S-layer homology proteins SLH (Bacillusanthraces, Anthrax); nucleocapsid protein NP, glycoprotein precursorGPC, glycoprotein GP1, glycoprotein GP2 (Junin virus, Argentinehemorrhagic fever); 41 kDa allergen Asp v13, allergen Asp f3, majorconidial surface protein rodlet A, protease Pep1p, GPI-anchored proteinGel1p, GPI-anchored protein Crf1p (Aspergillus genus, Aspergillosis);outer surface protein A OspA, outer surface protein OspB, outer surfaceprotein OspC, decorin binding protein A DbpA, flagellar filament 41 kDacore protein Fla, basic membrane protein A precursor BmpA(Immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor(antigen IPLA7), variable surface lipoprotein vIsE (Borrelia genus,Borrelia infection); OmpA-like transmembrane domain-containing proteinOmp31, immunogenic 39-kDa protein M5 P39, 25 kDa outer-membraneimmunogenic protein precursor Omp25, outer membrane protein MotY Omp16,conserved outer membrane protein D15, malate dehydrogenase Mdh,component of the Type-IV secretion system (T4SS) VirJ, lipoprotein ofunknown function BAB1_0187 (Brucella genus, Brucellosis); major outermembrane protein PorA, flagellin FIaA, surface antigen CjaA, fibronectinbinding protein CadF, aspartate/glutamate-binding ABC transporterprotein Peb1A, protein FspA1, protein FspA2 (Campylobacter genus,Campylobacteriosis); glycolytic enzyme enolase, secreted aspartylproteinases SAP1-10, glycophosphatidylinositol (GPI)-linked cell wallprotein, adhesin Als3p, cell surface hydrophobicity protein CSH (usuallyCandida albicans and other Candida species, Candidiasis); envelopeglycoproteins (gB, gC, gE, gH, gI, gK, gL) (Varicella zoster virus(VZV), Chickenpox); major outer membrane protein MOMP, probable outermembrane protein PMPC, outer membrane complex protein B OmcB (Chlamydiatrachomatis, Chlamydia); major outer membrane protein MOMP, outermembrane protein 2 Omp2, (Chlamydophila pneumoniae, Chlamydophilapneumoniae infection); outer membrane protein U Porin ompU, (Vibriocholerae, Cholera); surface layer proteins SLPs, Cell Wall Protein CwpV,flagellar protein FliC, flagellar protein FliD (Clostridium difficile,Clostridium difficile infection); acidic ribosomal protein P2 CpP2,mucin antigens Muc1, Muc2, Muc3 Muc4, Muc5, Muc6, Muc7, surfaceadherence protein CP20, surface adherence protein CP23, surface proteinCP12, surface protein CP21, surface protein CP40, surface protein CP60,surface protein CP15, surface-associated glycopeptides gp40,surface-associated glycopeptides gp15, oocyst wall protein AB, profilinPRF, apyrase (Cryptosporidium genus, Cryptosporidiosis); membraneprotein pp15, capsid-proximal tegument protein pp150 (Cytomegalovirus,Cytomegalovirus infection); prion protein (vCJD prion, VariantCreutzfeldt-Jakob disease (vCJD, nvCJD)); cyst wall proteins CWP1, CWP2,CWP3, variant surface protein VSP, VSP1, VSP2, VSP3, VSP4, VSP5, VSP6,56 kDa antigen (Giardia intestinalis, Giardiasis); minorpilin-associated subunit pilC, major pilin subunit and variants pilE,pilS (Neisseria gonorrhoeae, Gonorrhea); outer membrane protein A OmpA,outer membrane protein C OmpC, outer membrane protein K17 OmpK17(Klebsiella granulomatis, Granuloma inguinale (Donovanosis));fibronectin-binding protein Sfb (Streptococcus pyogenes, Group Astreptococcal infection); outer membrane protein P6 (Haemophilusinfluenzae, Haemophilus influenzae infection); integral membraneproteins, aggregation-prone proteins, O-antigen, toxin-antigens Stx2B,toxin-antigen Stx1B, adhesion-antigen fragment Int28, protein EspA,protein EspB, Intimin, protein Tir, protein IntC300, protein Eae(Escherichia coli O157:H7, O111 and O104:H4, Hemolytic-uremic syndrome(HUS)); hepatitis A surface antigen HBAg (Hepatitis A Virus, HepatitisA); hepatitis B surface antigen HBsAg (Hepatitis B Virus, Hepatitis B);envelope glycoprotein E1 gp32 gp35, envelope glycoprotein E2 NS1 gp68gp70, capsid protein C, (Hepatitis C Virus, Hepatitis C); type IV pilinPilE, outer membrane protein MIP, major outer membrane protein MompS(Legionella pneumophila, Legionellosis (Legionnaires' disease, Pontiacfever)); minor pilin-associated subunit pilC, major pilin subunit andvariants pilE, pilS (Neisseria meningitidis, Meningococcal disease);adhesin P1, adhesion P30 (Mycoplasma pneumoniae, Mycoplasma pneumonia);F1 capsule antigen, outer membrane protease Pla, (Yersinia pestis,Plague); surface adhesin PsaA, cell wall surface anchored protein psrP(Streptococcus pneumoniae, Pneumococcal infection); flagellin FliC,invasion protein SipC, glycoprotein gp43, outer membrane protein LamB,outer membrane protein PagC, outer membrane protein TolC, outer membraneprotein NmpC, outer membrane protein FadL, transport protein SadA(Salmonella genus, Salmonellosis); collagen adhesin Cna,fibronectin-binding protein A FnbA, secretory antigen SssA(Staphylococcus genus, Staphylococcal food poisoning); collagen adhesinCan (Staphylococcus genus, Staphylococcal infection);fibronectin-binding protein A FbpA (Ag85A), fibronectin-binding proteinD FbpD, fibronectin-binding protein C FbpC1, heat-shock protein HSP65,protein PST-S (Mycobacterium tuberculosis, Tuberculosis); and outermembrane protein FobA, outer membrane protein FobB, type IV piliglycosylation protein, outer membrane protein tolC, protein TolQ(Francisella tularensis, Tularemia). Additional exemplary microorganismsand corresponding antigens are disclosed, e.g., in U.S. Publication No.2015/0118264, the entire contents of which are expressly incorporatedherein by reference.

In some embodiments, a plurality of antibodies (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, or more antibodies) are used asanalyte-binding agents in any of the methods described herein (e.g., todetect the presence of one or more analytes in a sample). In someembodiments, the plurality of antibodies bind to the same analyte (e.g.,an antigen). In some embodiments, the plurality of antibodies bind tothe same epitope present on the analyte (e.g., an antigen). In someembodiments, the plurality of antibodies bind to different epitopespresent on the same analyte. In some embodiments, the plurality ofantibodies bind to overlapping epitopes present on the same analyte. Insome embodiments, the plurality of antibodies bind to non-overlappingepitopes present on the same analyte.

b. Antibiotics

In some embodiments, the analyte or analyte-binding agent is anantibiotic. An “antibiotic” or “antibiotic agent” refers to a substancethat has the capacity to inhibit or slow down the growth of, or todestroy bacteria and/or other microorganisms. In some embodiments, theantibiotic agent is a bacteriostatic antibiotic agent. In someembodiments, the antibiotic is a bacteriolytic antibiotic agent.Exemplary antibiotic agents are set forth in the U.S. Patent PublicationUS 2006/0269485, which is hereby incorporated by reference herein in itsentirety.

In some embodiments, the antibiotic agent is selected from the classesconsisting of beta-lactam antibiotics, aminoglycosides, ansa-typeantibiotics, anthraquinones, antibiotic azoles, antibioticglycopeptides, macrolides, antibiotic nucleosides, antibiotic peptides,antibiotic polyenes, antibiotic polyethers, quinolones, antibioticsteroids, sulfonamides, tetracycline, dicarboxylic acids, antibioticmetals, oxidizing agents, substances that release free radicals and/oractive oxygen, cationic antimicrobial agents, quaternary ammoniumcompounds, biguanides, triguanides, bisbiguanides and analogs andpolymers thereof and naturally occurring antibiotic compounds. In someembodiments, the antibiotic is rifaximin.

Beta-lactam antibiotics include, but are not limited to,2-(3-alanyl)clavam, 2-hydroxymethylclavam, 8-epi-thienamycin,acetyl-thienamycin, amoxicillin, amoxicillin sodium, amoxicillintrihydrate, amoxicillin-potassium clavulanate combination, ampicillin,ampicillin sodium, ampicillin trihydrate, ampicillin-sulbactam,apalcillin, aspoxicillin, azidocillin, azlocillin, aztreonam,bacampicillin, biapenem, carbenicillin, carbenicillin disodium,carfecillin, carindacillin, carpetimycin, cefacetril, cefaclor,cefadroxil, cefalexin, cefaloridine, cefalotin, cefamandole,cefamandole, cefapirin, cefatrizine, cefatrizine propylene glycol,cefazedone, cefazolin, cefbuperazone, cefcapene, cefcapene pivoxilhydrochloride, cefdinir, cefditoren, cefditoren pivoxil, cefepime,cefetamet, cefetamet pivoxil, cefixime, cefinenoxime, cefinetazole,cefminox, cefminox, cefmolexin, cefodizime, cefonicid, cefoperazone,ceforanide, cefoselis, cefotaxime, cefotetan, cefotiam, cefoxitin,cefozopran, cefpiramide, cefpirome, cefpodoxime, cefpodoxime proxetil,cefprozil, cefquinome, cefradine, cefroxadine, cefsulodin, ceftazidime,cefteram, cefteram pivoxil, ceftezole, ceftibuten, ceftizoxime,ceftriaxone, cefuroxime, cefuroxime axetil, cephalosporin, cephamycin,chitinovorin, ciclacillin, clavulanic acid, clometocillin, cloxacillin,cycloserine, deoxy pluracidomycin, dicloxacillin, dihydropluracidomycin, epicillin, epithienamycin, ertapenem, faropenem,flomoxef, flucloxacillin, hetacillin, imipenem, lenampicillin,loracarbef, mecillinam, meropenem, metampicillin, meticillin,mezlocillin, moxalactam, nafcillin, northienamycin, oxacillin,panipenem, penamecillin, penicillin, phenethicillin, piperacillin,tazobactam, pivampicillin, pivcefalexin, pivmecillinam, pivmecillinamhydrochloride, pluracidomycin, propicillin, sarmoxicillin, sulbactam,sulbenicillin, talampicillin, temocillin, terconazole, thienamycin,ticarcillin and analogs, salts and derivatives thereof.

Aminoglycosides include, but are not limited to,1,2′-N-DL-isoseryl-3′,4′-dideoxykanamycin B,1,2′-N-DL-isoseryl-kanamycin B,1,2′-N—[(S)-4-amino-2-hydroxybutyryl]-3′,4′-dideoxykanamycin B,1,2′-N—[(S)-4-amino-2-hydroxybutyryl]-kanamycin B,1-N-(2-Aminobutanesulfonyl) kanamycin A,1-N-(2-aminoethanesulfonyl)3′,4′-dideoxyribostamycin,1-N-(2-Aminoethanesulfonyl)3′-deoxyribostamycin,1-N-(2-aminoethanesulfonyl)3′4′-dideoxykanamycin B,1-N-(2-aminoethanesulfonyl)kanamycin A,1-N-(2-aminoethanesulfonyl)kanamycin B,1-N-(2-aminoethanesulfonyl)ribostamycin,1-N-(2-aminopropanesulfonyl)3′-deoxykanamycin B,1-N-(2-aminopropanesulfonyl)3′4′-dideoxykanamycin B,1-N-(2-aminopropanesulfonyl)kanamycin A,1-N-(2-aminopropanesulfonyl)kanamycin B,1-N-(L-4-amino-2-hydroxy-butyryl)2,′3′-dideoxy-2′-fluorokanamycin A,1-N-(L-4-amino-2-hydroxy-propionyl)2,′3′-dideoxy-2′-fluorokanamycin A,1-N-DL-3′,4′-dideoxy-isoserylkanamycin B, 1-N-DL-isoserylkanamycin,1-N-DL-isoserylkanamycin B,1-N-[L-(−)-(alpha-hydroxy-gamma-aminobutyryl)]-XK-62-2,2′,3′-dideoxy-2′-fluorokanamycinA, 2-hydroxygentamycin A3,2-hydroxygentamycin B, 2-hydroxygentamycin B1,2-hydroxygentamycin JI-20A, 2-hydroxygentamycin3″-N-methyl-4″-C-methyl-3′,4′-dodeoxy kanamycin A,3″-N-methyl-4″-C-methyl-3′,4′-dodeoxy kanamycin B,3″-N-methyl-4″-C-methyl-3′,4′-dodeoxy-6′-methylkanamycin B,3′,4′-Dideoxy-3′-eno-ribostamycin, 3′,4′-dideoxyneamine,3′,4′-dideoxyribostamycin, 3′-deoxy-6′-N-methyl-kanamycin B,3′-deoxyneamine, 3′-deoxyribostamycin, 3′-oxysaccharocin,3,3′-nepotrehalosadiamine, 3-demethoxy-2″-N-formimidoylistamycin Bdisulfate tetrahydrate, 3-demethoxyistamycin B,3-O-demethyl-2-N-formimidoylistamycin B, 3-O-demethylistamycin B,3-trehalosamine, 4″,6″-dideoxydibekacin, 4-N-glycyl-KA-6606VI,5″-Amino-3′,4′,5″-trideoxy-butirosin A, 6″-deoxydibekacin,6′-epifortimicin A, 6-deoxy-neomycin (structure 6-deoxy-neomycin B),6-deoxy-neomycin B, 6-deoxy-neomycin C, 6-deoxy-paromomycin, acmimycin,AHB-3′,4′-dideoxyribostamycin, AHB-3′-deoxykanamycin B,AHB-3′-deoxyneamine, AHB-3′-deoxyribostamycin,AHB-4″-6″-dideoxydibekacin, AHB-6″-deoxydibekacin, AHB-dideoxyneamine,AHB-kanamycin B, AHB-methyl-3′-deoxykanamycin B, amikacin, amikacinsulfate, apramycin, arbekacin, astromicin, astromicin sulfate,bekanamycin, bluensomycin, boholmycin, butirosin, butirosin B,catenulin, coumamidine gamma1, coumamidinegamma2,D,L-1-N-(alpha-hydroxy-beta-aminopropionyl)-XK-62-2, dactimicin,de-O-methyl-4-N-glycyl-KA-6606VI, de-O-methyl-KA-66061,de-O-methyl-KA-7038I, destomycin A, destomycin B,di-N6′,O3-demethylistamycin A, dibekacin, dibekacin sulfate,dihydrostreptomycin, dihydrostreptomycin sulfate,epi-formamidoylglycidylfortimicin B, epihygromycin,formimidoyl-istamycin A, formimidoyl-istamycin B, fortimicin B,fortimicin C, fortimicin D, fortimicin KE, fortimicin KF, fortimicin KG,fortimicin KG1 (stereoisomer KG1/KG2), fortimicin KG2 (stereoisomerKG1/KG2), fortimicin KG3, framycetin, framycetin sulphate, gentamicin,gentamycin sulfate, globeomycin, hybrimycin A1, hybrimycin A2,hybrimycin B1, hybrimycin B2, hybrimycin C1, hybrimycin C2,hydroxystreptomycin, hygromycin, hygromycin B, isepamicin, isepamicinsulfate, istamycin, kanamycin, kanamycin sulphate, kasugamycin,lividomycin, marcomycin, micronomicin, micronomicin sulfate, mutamicin,myomycin, N-demethyl-7-O-demethylcelesticetin, demethylcelesticetin,methanesulfonic acid derivative of istamycin, nebramycin, nebramycin,neomycin, netilmicin, oligostatin, paromomycin, quintomycin,ribostamycin, saccharocin, seldomycin, sisomicin, sorbistin,spectinomycin, streptomycin, tobramycin, trehalosmaine, trestatin,validamycin, verdamycin, xylostasin, zygomycin and analogs, salts andderivatives thereof.

Ansa-type antibiotics include, but are not limited to,21-hydroxy-25-demethyl-25-methylth ioprotostreptovaricin, 3-methylthiorifamycin, ansamitocin, atropisostreptovaricin, awamycin, halomicin,maytansine, naphthomycin, rifabutin, rifamide, rifampicin, rifamycin,rifapentine, rifaximin (e.g., Xifaxan®), rubradirin, streptovaricin,tolypomycin and analogs, salts and derivatives thereof.

Antibiotic anthraquinones include, but are not limited to, auramycin,cinerubin, ditrisarubicin, ditrisarubicin C, figaroic acid fragilomycin,minomycin, rabelomycin, rudolfomycin, sulfurmycin and analogs, salts andderivatives thereof.

Antibiotic azoles include, but are not limited to, azanidazole,bifonazole, butoconazol, chlormidazole, chlormidazole hydrochloride,cloconazole, cloconazole monohydrochloride, clotrimazol, dimetridazole,econazole, econazole nitrate, enilconazole, fenticonazole, fenticonazolenitrate, fezatione, fluconazole, flutrimazole, isoconazole, isoconazolenitrate, itraconazole, ketoconazole, lanoconazole, metronidazole,metronidazole benzoate, miconazole, miconazole nitrate, neticonazole,nimorazole, niridazole, omoconazol, ornidazole, oxiconazole, oxiconazolenitrate, propenidazole, secnidazol, sertaconazole, sertaconazolenitrate, sulconazole, sulconazole nitrate, tinidazole, tioconazole,voriconazol and analogs, salts and derivatives thereof.

Antibiotic glycopeptides include, but are not limited to, acanthomycin,actaplanin, avoparcin, balhimycin, bleomycin B (copper bleomycin),chloroorienticin, chloropolysporin, demethylvancomycin, enduracidin,galacardin, guanidylfungin, hachimycin, demethylvancomycin,N-nonanoyl-teicoplanin, phleomycin, platomycin, ristocetin,staphylocidin, talisomycin, teicoplanin, vancomycin, victomycin,xylocandin, zorbamycin and analogs, salts and derivatives thereof.

Macrolides include, but are not limited to, acetylleucomycin,acetylkitasamycin, angolamycin, azithromycin, bafilomycin, brefeldin,carbomycin, chalcomycin, cirramycin, clarithromycin, concanamycin,deisovaleryl-niddamycin, demycinosyl-mycinamycin,Di-O-methyltiacumicidin, dirithromycin, erythromycin, erythromycinestolate, erythromycin ethyl succinate, erythromycin lactobionate,erythromycin stearate, flurithromycin, focusin, foromacidin,haterumalide, haterumalide, josamycin, josamycin ropionate, juvenimycin,juvenimycin, kitasamycin, ketotiacumicin, lankavacidin, lankavamycin,leucomycin, machecin, maridomycin, megalomicin, methylleucomycin,methymycin, midecamycin, miocamycin, mycaminosyltylactone, mycinomycin,neutramycin, niddamycin, nonactin, oleandomycin, phenylacetyideltamycin,pamamycin, picromycin, rokitamycin, rosaramicin, roxithromycin,sedecamycin, shincomycin, spiramycin, swalpamycin, tacrolimus,telithromycin, tiacumicin, tilmicosin, treponemycin, troleandomycin,tylosin, venturicidin and analogs, salts and derivatives thereof.

Antibiotic nucleosides include, but are not limited to, amicetin,angustmycin, azathymidine, blasticidin S, epiroprim, flucytosine,gougerotin, mildiomycin, nikkomycin, nucleocidin, oxanosine, oxanosine,puromycin, pyrazomycin, showdomycin, sinefungin, sparsogenin,spicamycin, tunicamycin, uracil polyoxin, vengicide and analogs, saltsand derivatives thereof.

Antibiotic peptides include, but are not limited to, actinomycin,aculeacin, alazopeptin, amfomycin, amythiamycin, antifungal fromZalerion arboricola, antrimycin, apid, apidaecin, aspartocin,auromomycin, bacileucin, bacillomycin, bacillopeptin, bacitracin,bagacidin, beminamycin, beta-alanyl-L-tyrosine, bottromycin,capreomycin, caspofungine, cepacidine, cerexin, cilofungin, circulin,colistin, cyclodepsipeptide, cytophagin, dactinomycin, daptomycin,decapeptide, desoxymulundocandin, echanomycin, echinocandin B,echinomycin, ecomycin, enniatin, etamycin, fabatin, ferrimycin,ferrimycin, ficellomycin, fluoronocathiacin, fusaricidin, gardimycin,gatavalin, globopeptin, glyphomycin, gramicidin, herbicolin, iomycin,iturin, iyomycin, izupeptin, janiemycin, janthinocin, jolipeptin,katanosin, killertoxin, lipopeptide antibiotic, lipopeptide fromZalerion sp., lysobactin, lysozyme, macromomycin, magainin, melittin,mersacidin, mikamycin, mureidomycin, mycoplanecin, mycosubtilin,neopeptifluorin, neoviridogrisein, netropsin, nisin, nocathiacin,nocathiacin 6-deoxyglycoside, nosiheptide, octapeptin, pacidamycin,pentadecapeptide, peptifluorin, permetin, phytoactin, phytostreptin,planothiocin, plusbacin, polcillin, polymyxin antibiotic complex,polymyxin B, polymyxin B1, polymyxin F, preneocarzinostatin, quinomycin,quinupristin-dalfopristin, safracin, salmycin, salmycin, salmycin,sandramycin, saramycetin, siomycin, sperabillin, sporamycin, aStreptomyces compound, subtilin, teicoplanin aglycone, telomycin,thermothiocin, thiopeptin, thiostrepton, tridecaptin, tsushimycin,tuberactinomycin, tuberactinomycin, tyrothricin, valinomycin, viomycin,virginiamycin, zervacin and analogs, salts and derivatives thereof.

In some embodiments, the antibiotic peptide is a naturally-occurringpeptide that possesses an antibacterial and/or an antifungal activity.Such peptide can be obtained from an herbal or a vertebrate source.

Polyenes include, but are not limited to, amphotericin, amphotericin,aureofungin, ayfactin, azalomycin, blasticidin, candicidin, candicidinmethyl ester, candimycin, candimycin methyl ester, chinopricin, filipin,flavofungin, fradicin, hamycin, hydropricin, levorin, lucensomycin,lucknomycin, mediocidin, mediocidin methyl ester, mepartricin,methylamphotericin, natamycin, niphimycin, nystatin, nystatin methylester, oxypricin, partricin, pentamycin, perimycin, pimaricin, primycin,proticin, rimocidin, sistomycosin, sorangicin, trichomycin and analogs,salts and derivatives thereof.

Polyethers include, but are not limited to, 20-deoxy-epi-narasin,20-deoxysalinomycin, carriomycin, dianemycin, dihydrolonomycin,etheromycin, ionomycin, iso-lasalocid, lasalocid, lenoremycin,lonomycin, lysocellin, monensin, narasin, oxolonomycin, a polycyclicether antibiotic, salinomycin and analogs, salts and derivativesthereof.

Quinolones include, but are not limited to, analkyl-methylendioxy-4(1H)-oxocinnoline-3-carboxylic acid,alatrofloxacin, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride,danofloxacin, dermofongin A, enoxacin, enrofloxacin, fleroxacin,flumequine, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin,lomefloxacin, lomefloxacin, hydrochloride, miloxacin, moxifloxacin,nadifloxacin, nalidixic acid, nifuroquine, norfloxacin, ofloxacin,orbifloxacin, oxolinic acid, pazufloxacine, pefloxacin, pefloxacinmesylate, pipemidic acid, piromidic acid, premafloxacin, rosoxacin,rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin andanalogs, salts and derivatives thereof.

Antibiotic steroids include, but are not limited to, aminosterol,ascosteroside, cladosporide A, dihydrofusidic acid,dehydro-dihydrofusidic acid, dehydrofusidic acid, fusidic acid,squalamine and analogs, salts and derivatives thereof.

Sulfonamides include, but are not limited to, chloramine, dapsone,mafenide, phthalylsulfathiazole, succinylsulfathiazole, sulfabenzamide,sulfacetamide, sulfachlorpyridazine, sulfadiazine, sulfadiazine silver,sulfadicramide, sulfadimethoxine, sulfadoxine, sulfaguanidine,sulfalene, sulfamazone, sulfamerazine, sulfamethazine, sulfamethizole,sulfamethoxazole, sulfamethoxypyridazine, sulfamonomethoxine,sulfamoxol, sulfanilamide, sulfaperine, sulfaphenazol, sulfapyridine,sulfaquinoxaline, sulfasuccinamide, sulfathiazole, sulfathiourea,sulfatolamide, sulfatriazin, sulfisomidine, sulfisoxazole, sulfisoxazoleacetyl, sulfacarbamide and analogs, salts and derivatives thereof.

Tetracyclines include, but are not limited to, dihydrosteffimycin,demethyltetracycline, aclacinomycin, akrobomycin, baumycin,bromotetracycline, cetocyclin, chlortetracycline, clomocycline,daunorubicin, demeclocycline, doxorubicin, doxorubicin hydrochloride,doxycycline, lymecyclin, marcellomycin, meclocycline, meclocyclinesulfosalicylate, methacycline, minocycline, minocycline hydrochloride,musettamycin, oxytetracycline, rhodirubin, rolitetracycline, rubomycin,serirubicin, steffimycin, tetracycline and analogs, salts andderivatives thereof.

Dicarboxylic acids, having between about 6 and about 14 carbon atoms intheir carbon atom skeleton are particularly useful in the treatment ofdisorders of the skin and mucosal membranes that involve microbial.Suitable dicarboxylic acid moieties include, but are not limited to,adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid,1,11-undecanedioic acid, 1,12-dodecanedioic acid, 1,13-tridecanedioicacid and 1,14-tetradecanedioic acid. Thus, in one or more embodiments ofthe present disclosure, dicarboxylic acids, having between about 6 andabout 14 carbon atoms in their carbon atom skeleton, as well as theirsalts and derivatives (e.g., esters, amides, mercapto-derivatives,anhydraides), are useful immunomodulators in the treatment of disordersof the skin and mucosal membranes that involve inflammation. Azelaicacid and its salts and derivatives are preferred. It has antibacterialeffects on both aerobic and anaerobic organisms, particularlyPropionibacterium acnes and Staphylococcus epidermidis, normalizeskeratinization, and has a cytotoxic effect on malignant or hyperactivemelanocytes. In a preferred embodiment, the dicarboxylic acid is azelaicacid in a concentration greater than 10%. Preferably, the concentrationof azelaic acid is between about 10% and about 25%. In suchconcentrates, azelaic acid is suitable for the treatment of a variety ofskin disorders, such as acne, rosacea and hyperpigmentation.

In some embodiments, the antibiotic agent is an antibiotic metal. Anumber of metals ions have been shown to possess antibiotic activity,including silver, copper, zinc, mercury, tin, lead, bismutin, cadmium,chromium and ions thereof. It has been theorized that these antibioticmetal ions exert their effects by disrupting respiration and electrontransport systems upon absorption into bacterial or fungal cells.Anti-microbial metal ions of silver, copper, zinc, and gold, inparticular, are considered safe for in vivo use. Anti-microbial silverand silver ions are particularly useful due to the fact that they arenot substantially absorbed into the body. Thus, in one or moreembodiment, the antibiotic metal consists of an elemental metal,selected from the group consisting of silver, copper, zinc, mercury,tin, lead, bismutin, cadmium, chromium and gold, which is suspended inthe composition as particles, microparticles, nanoparticles or colloidalparticles. The antibiotic metal can further be intercalated in achelating substrate.

In further embodiments, the antibiotic metal is ionic. The ionicantibiotic metal can be presented as an inorganic or organic salt(coupled with a counterion), an organometallic complex or anintercalate. Non-binding examples of counter inorganic and organic ionsare sulfadiazine, acetate, benzoate, carbonate, iodate, iodide, lactate,laurate, nitrate, oxide, and palmitate, a negatively charged protein. Inpreferred embodiments, the antibiotic metal salt is a silver salt, suchas silver acetate, silver benzoate, silver carbonate, silver iodate,silver iodide, silver lactate, silver laurate, silver nitrate, silveroxide, silver palmitate, silver protein, and silver sulfadiazine.

In one or more embodiments, the antibiotic metal or metal ion isembedded into a substrate, such as a polymer, or a mineral (such aszeolite, clay and silica).

In one or more embodiments, the antibiotic agent includes strongoxidants and free radical liberating compounds, such as oxygen, hydrogenperoxide, benzoyl peroxide, elemental halogen species, as well asoxygenated halogen species, bleaching agents (e.g., sodium, calcium ormagnesium hypochloride and the like), perchlorite species, iodine,iodate, and benzoyl peroxide. Organic oxidizing agents, such asquinones, are also included. Such agents possess a potent broad-spectrumactivity.

In one or more embodiments, the antibiotic agent is a cationicantimicrobial agent. The outermost surface of bacterial cellsuniversally carries a net negative charge, making them sensitive tocationic substances. Examples of cationic antibiotic agents include:quaternary ammonium compounds (QAC's)—QAC's are surfactants, generallycontaining one quaternary nitrogen associated with at least one majorhydrophobic moiety; alkyltrimethyl ammonium bromides are mixtures ofwhere the alkyl group is between 8 and 18 carbons long, such ascetrimide (tetradecyltrimethylammonium bromide); benzalkonium chloride,which is a mixture of n-alkyldimethylbenzyl ammonium chloride where thealkyl groups (the hydrophobic moiety) can be of variable length;dialkylmethyl ammonium halides; dialkylbenzyl ammonium halides; and QACdimmers, which bear bi-polar positive charges in conjunction withinterstitial hydrophobic regions.

In one or more embodiments, the cationic antimicrobial agent is apolymer. Cationic antimicrobial polymers include, for example, guanidepolymers, biguanide polymers, or polymers having side chains containingbiguanide moieties or other cationic functional groups, such asbenzalkonium groups or quartemium groups (e.g., quaternary aminegroups). It is understood that the term “polymer” as used hereinincludes any organic material including three or more repeating units,and includes oligomers, polymers, copolymers, block copolymers,terpolymers, etc. The polymer backbone may be, for example apolyethylene, ploypropylene or polysilane polymer.

In one or more embodiments, the cationic antimicrobial polymer is apolymeric biguanide compound. When applied to a substrate, such apolymer is known to form a barrier film that can engage and disrupt amicroorganism. An exemplary polymeric biguanide compound ispolyhexamethylene biguanide (PHMB) salts. Other exemplary biguanidepolymers include, but are not limited to poly(hexamethylenebiguanide),poly(hexamethylenebiguanide) hydrochloride, poly(hexamethylenebiguanide)gluconate, poly(hexamethylenebiguanide) stearate, or a derivativethereof. In one or more embodiments, the antimicrobial material issubstantially water-insoluble.

In some embodiments, the antibiotic agent is selected from the group ofbiguanides, triguanides, bisbiguanides and analogs thereof.

Guanides, biguanides, biguanidines and triguanides are unsaturatednitrogen containing molecules that readily obtain one or more positivecharges, which make them effective antimicrobial agents. The basicstructures a guanide, a biguanide, a biguanidine and a triguanide areprovided below.

In some embodiments, the guanide, biguanide, biguanidine or triguanide,provide bi-polar configurations of cationic and hydrophobic domainswithin a single molecule.

Examples of guanides, biguanides, biguanidines and triguanides that arecurrently been used as antibacterial agents include chlorhexidine andchlorohexidine salts, analogs and derivatives, such as chlorhexidineacetate, chlorhexidine gluconate and chlorhexidine hydrochloride,picloxydine, alexidine and polihexanide. Other examples of guanides,biguanides, biguanidines and triguanides that can conceivably be usedaccording to the present disclosure are chlorproguanil hydrochloride,proguanil hydrochloride (currently used as antimalarial agents),mefformin hydrochloride, phenformin and buformin hydrochloride(currently used as antidiabetic agents).

Yet, in one or more embodiments, the antibiotic is a non-classifiedantibiotic agent, including, without limitation, aabomycin, acetomycin,acetoxycycloheximide, acetylnanaomycin, an Actinoplanes sp. compound,actinopyrone, aflastatin, albacarcin, albacarcin, albofungin,albofungin, alisamycin, alpha-R,S-methoxycarbonylbenzylmonate,altromycin, amicetin, amycin, amycin demanoyl compound, amycine,amycomycin, anandimycin, anisomycin, anthramycin, anti-syphilis immunesubstance, anti-tuberculosis immune substance, an antibiotic fromEscherichia coli, an antibiotic from Streptomyces refuineus, anticapsin,antimycin, aplasmomycin, aranorosin, aranorosinol, arugomycin,ascofuranone, ascomycin, ascosin, Aspergillus flavus antibiotic,asukamycin, aurantinin, an Aureolic acid antibiotic substance, aurodox,avilamycin, azidamfenicol, azidimycin, bacillaene, a Bacillus larvaeantibiotic, bactobolin, benanomycin, benzanthrin, benzylmonate,bicozamycin, bravomicin, brodimoprim, butalactin, calcimycin, calvaticacid, candiplanecin, carumonam, carzinophilin, celesticetin, cepacin,cerulenin, cervinomycin, chartreusin, chloramphenicol, chloramphenicolpalmitate, chloramphenicol succinate sodium, chlorflavonin,chlorobiocin, chlorocarcin, chromomycin, ciclopirox, ciclopirox olamine,citreamicin, cladosporin, clazamycin, clecarmycin, clindamycin,coliformin, collinomycin, copiamycin, corallopyronin, corynecandin,coumermycin, culpin, cuprimyxin, cyclamidomycin, cycloheximide,dactylomycin, danomycin, danubomycin, delaminomycin, demethoxyrapamycin,demethylscytophycin, dermadin, desdamethine, dexylosyl-benanomycin,pseudoaglycone, dihydromocimycin, dihydronancimycin, diumycin, dnacin,dorrigocin, dynemycin, dynemycin triacetate, ecteinascidin, efrotomycin,endomycin, ensanchomycin, equisetin, ericamycin, esperamicin,ethylmonate, everninomicin, feldamycin, flambamycin, flavensomycin,florfenicol, fluvomycin, fosfomycin, fosfonochlorin, fredericamycin,frenolicin, fumagillin, fumifungin, funginon, fusacandin, fusafungin,gelbecidine, glidobactin, grahamimycin, granaticin, griseofulvin,griseoviridin, grisonomycin, hayumicin, hayumicin, hazymicin, hedamycin,heneicomycin, heptelicid acid, holomycin, humidin, isohematinic acid,karnatakin, kazusamycin, kristenin, L-dihydrophenylalanine, aL-isoleucyl-L-2-amino-4-(4′-amino-2′,5′-cyclohexadienyl) derivative,lanomycin, leinamycin, leptomycin, libanomycin, lincomycin, lomofungin,lysolipin, magnesidin, manumycin, melanomycin,methoxycarbonylmethylmonate, methoxycarbonylethylmonate,methoxycarbonylphenylmonate, methyl pseudomonate, methylmonate,microcin, mitomalcin, mocimycin, moenomycin, monoacetyl cladosporin,monomethyl cladosporin, mupirocin, mupirocin calcium, mycobacidin,myriocin, myxopyronin, pseudoaglycone, nanaomycin, nancimycin,nargenicin, neocarcinostatin, neoenactin, neothramycin, nifurtoinol,nocardicin, nogalamycin, novobiocin, octylmonate, olivomycin,orthosomycin, oudemansin, oxirapentyn, oxoglaucine methiodide, pactacin,pactamycin, papulacandin, paulomycin, phaeoramularia fungicide,phenelfamycin, phenyl, cerulenin, phenylmonate, pholipomycin,pirlimycin, pleuromutilin, a polylactone derivative, polynitroxin,polyoxin, porfiromycin, pradimicin, prenomycin, prop-2-enylmonate,protomycin, Pseudomonas antibiotic, pseudomonic acid, purpuromycin,pyrinodemin, pyrrolnitrin, pyrrolomycin, amino, chloro pentenedioicacid, rapamycin, rebeccamycin, resistomycin, reuterin, reveromycin,rhizocticin, roridin, rubiflavin, naphthyridinomycin, saframycin,saphenamycin, sarkomycin, sarkomycin, sclopularin, selenomycin,siccanin, spartanamicin, spectinomycin, spongistatin, stravidin,streptolydigin, Streptomyces arenae antibiotic complex, streptonigrin,streptothricins, streptovitacin, streptozotocine, a strobilurinderivative, stubomycin, sulfamethoxazol-trimethoprim, sakamycin,tejeramycin, terpentecin, tetrocarcin, thermorubin, thermozymocidin,thiamphenicol, thioaurin, thiolutin, thiomarinol, thiomarinol,tirandamycin, tolytoxin, trichodermin, trienomycin, trimethoprim,trioxacarcin, tyrissamycin, umbrinomycin, unphenelfamycin, urauchimycin,usnic acid, uredolysin, variotin, vermisporin, verrucarin and analogs,salts and derivatives thereof.

In one or more embodiments, the antibiotic agent is a naturallyoccurring antibiotic compound. As used herein, the term“naturally-occurring antibiotic agent” includes all antibiotics that areobtained, derived or extracted from plant or vertebrate sources.Non-limiting examples of families of naturally-occurring antibioticagents include phenol, resorcinol, antibiotic aminoglycosides, anamycin,quinines, anthraquinones, antibiotic glycopeptides, azoles, macrolides,avilamycin, agropyrene, cnicin, aucubin antibioticsaponin fractions,berberine (isoquinoline alkaloid), arctiopicrin (sesquiterpene lactone),lupulone, humulone (bitter acids), allicin, hyperforin, echinacoside,coniosetin, tetramic acid, imanine and novoimanine.

Ciclopirox and ciclopiroxolamine possess fungicidal, fungistatic andsporicidal activity. They are active against a broad spectrum ofdermatophytes, yeasts, moulds and other fungi, such as Trichophytonsspecies, Microsporum species, Epidermophyton species and yeasts (Candidaalbicans, Candida glabrata, other candida species and Cryptococcusneoformans). Some Aspergillus species are sensitive to ciclopirox as aresome Penicillium. Likewise, ciclopirox is effective against manyGram-positive and Gram-negative bacteria (e.g., Escherichia coli,Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus andStreptococcus species), as well as Mycoplasma species, Trichomonasvaginalis and Actinomyces.

Plant oils and extracts which contain antibiotic agents are also useful.Non-limiting examples of plants that contain agents include thyme,Perilla, lavender, tea tree, Terfezia clayeryi, Micromonospora,Putterlickia verrucosa, Putterlickia pyracantha, Putterlickiaretrospinosa, Maytenus ilicifolia, Maytenus evonymoides, Maytenusaquifolia, Faenia interjecta, Cordyceps sinensis, couchgrass, holythistle, plantain, burdock, hops, echinacea, buchu, chaparral, myrrh,red clover and yellow dock, garlic, and St. John's wort. Mixtures of theantibiotic agents as described herein may also be employed.

Combination Detection:

Any combination of the analytes disclosed herein can be detected usingany of the methods described herein. In particular, any combinationdisclosed herein can be detected using any of the methods describedherein.

A “photosensitizer” as used herein refers to a sensitizer for generationof singlet oxygen usually by excitation with light. Exemplaryphotosensitizers suitable for use include those described in U.S. Pat.Nos. 6,251,581, 5,516,636, 8,907,081, 6,545,012, 6,331,530, 8,247,180,5,763,602, 5,705,622, 5,516,636, 7,217,531, and U.S. Patent PublicationNo. 2007/0059316, all of which are herein expressly incorporated byreference in their entireties. The photosensitizer can bephotoactivatable (e.g., dyes and aromatic compounds) or chemiactivated(e.g., enzymes and metal salts). When excited by light thephotosensitizer is usually a compound comprised of covalently bondedatoms, usually with multiple conjugated double or triple bonds. Thecompound should absorb light in the wavelength range of 200-1100 nm,usually 300-1000 nm, e.g., 450-950 nm, with an extinction coefficient atits absorbance maximum greater than 500 M⁻¹cm⁻¹, e.g., at least 5000M⁻¹cm⁻¹, or at least 50,000 M⁻¹cm⁻¹ at the excitation wavelength. Thelifetime of an excited state produced following absorption of light inthe absence of oxygen will usually be at least 100 nsec, e.g., at least1 μsec. In general, the lifetime must be sufficiently long to permitenergy transfer to oxygen, which will normally be present atconcentrations in the range of 10⁻⁵ to 10³¹ ³M depending on the medium.The sensitizer excited state will usually have a different spin quantumnumber (S) than its ground state and will usually be a triplet (S=1)when, as is usually the case, the ground state is a singlet (S═O). Insome embodiments, the sensitizer will have a high intersystem crossingyield. That is, photoexcitation of a sensitizer will produce the longlived state (usually triplet) with an efficiency of at least 10%, atleast 40%, e.g., greater than 80%. The photosensitizer will usually beat most weakly fluorescent under the assay conditions (quantum yieldusually less that 0.5, or less that 0.1).

Photosensitizers that are to be excited by light will be relativelyphotostable and will not react efficiently with singlet oxygen. Severalstructural features are present in most useful sensitizers. Mostsensitizers have at least one and frequently three or more conjugateddouble or triple bonds held in a rigid, frequently aromatic structure.They will frequently contain at least one group that acceleratesintersystem crossing such as a carbonyl or imine group or a heavy atomselected from rows 3-6 of the periodic table, especially iodine orbromine, or they may have extended aromatic structures. Typicalsensitizers include acetone, benzophenone, 9-thioxanthone, eosin,9,10-dibromoanthracene, methylene blue, metallo-porphyrins, such ashematoporphyrin, phthalocyanines, chlorophylls, rose bengal,buckminsterfullerene, etc., and derivatives of these compounds havingsubstituents of 1 to 50 atoms for rendering such compounds morelipophilic or more hydrophilic and/or as attaching groups forattachment. Examples of other photosensitizers that may be utilized arethose that have the above properties and are enumerated in N. J. Turro,“Molecular Photochemistry,” page 132, W. A. Benjamin Inc., N.Y. 1965.

In some embodiments, the photosensitizers are relatively non-polar toassure dissolution into a lipophilic member when the photosensitizer isincorporated in an oil droplet, liposome, latex particle, etc.

In some embodiments, the photosensitizers suitable for use hereininclude other substances and compositions that can produce singletoxygen with or without activation by an external light source. Thus, forexample, molybdate (MoO₄ ⁻) salts and chloroperoxidase andmyeloperoxidase plus bromide or chloride ion (Kanofsky, J. Biol. Chem.(1983) 259 5596) have been shown to catalyze the conversion of hydrogenperoxide to singlet oxygen and water. Either of these compositions can,for example, be included in particles and used in the assay methodwherein hydrogen peroxide is included as an ancillary reagebly,chloroperoxidase is bound to a surface and molybdate is incorporated inthe aqueous phase of a liposome. Also included within the scope of theinvention as photosensitizers are compounds that are not truesensitizers but which on excitation by heat, light, or chemicalactivation will release a molecule of singlet oxygen. The best knownmembers of this class of compounds includes the endoperoxides such as1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen.

A “chemiluminescent compound” as used herein refers to a substance thatundergoes a chemical reaction with singlet oxygen to form a metastableintermediate that can decompose with the simultaneous or subsequentemission of light within the wavelength range of 250 to 1200 nm.Exemplary chemiluminescent compounds suitable for use include thosedescribed in U.S. Pat. Nos. 6,251,581 and 7,709,273, and PatentCooperatio Treaty (PCT) International Application Publication No.WO1999/042838. Exemplary chemiluminescent compound includes thefollowing:

Chemiluminescer Half-Life Emission Max Thioxene + Diphenyl anthracence:0.6 seconds 430 nm Thioxene + Umbelliferone derivative 0.6 seconds 500nm Thioxene + Europium chelate 0.6 seconds 615 nm Thioxene + SamariumChelate 0.6 seconds 648 nm Thioxene + terbium Chelate 0.6 seconds 540 nmN-Phenyl Oxazine + Umbelliferone  30 seconds 500 nm derivative N-PhenylOxazine + Europium chelate  30 seconds 613 nm N-phenyl Oxazine +Samarium Chelate  30 seconds 648 nm N-phenyl Oxazine + terbium Chelate 30 seconds 540 nm Dioxene + Umbelliferone derivative 300 seconds  500nm Dioxene + Europium chelate 300 seconds  613 nm Dioxene + SamariumChelate 300 seconds  648 nm N-phenyl Oxazine + terbium Chelate 300seconds  540 nm

All of the above mentioned applications are hereby expresslyincorporated by reference herein in their entireties. Emission willusually occur without the presence of an energy acceptor or catalyst tocause decomposition and light emission. In some embodiments, theintermediate decomposes spontaneously without heating or addition ofancillary reagents following its formation. However, addition of areagent after formation of the intermediate or the use of elevatedtemperature to accelerate decomposition will be required for somechemiluminescent compounds. The chemiluminescent compounds are usuallyelectron rich compounds that react with singlet oxygen, frequently withformation of dioxetanes or dioxetanones. Exemplary of such compounds areenol ethers, enamines, 9-alkylidenexanthans,9-alkylidene-N-alkylacridans, aryl vinyl ethers, dioxenes,arylimidazoles and lucigenin. Other chemiluminescent compounds giveintermediates upon reaction with singlet oxygen, which subsequentlyreact with another reagent with light emission. Exemplary compounds arehydrazides such as luminol and oxalate esters.

The chemiluminescent compounds of interest will generally emit atwavelengths above 300 nanometers and usually above 400 nm. Compoundsthat alone or together with a fluorescent molecule emit light atwavelengths beyond the region where serum components absorb light willbe of particular use. The fluorescence of serum drops off rapidly above500 nm and becomes relatively unimportant above 550 nm. Therefore, whenthe analyte is in serum, chemiluminescent compounds that emit lightabove 550 nm, e.g., above 600 nm may be suitable for use. In order toavoid autosensitization of the chemiluminescent compound, in someembodiments, the chemiluminescent compounds do not absorb light used toexcite the photosensitizer. In some embodiments, the sensitizer isexcited with light wavelengths longer than 500 nm, it will therefore bedesirable that light absorption by the chemiluminescent compound be verylow above 500 nm.

Where long wave length emission from the chemiluminescent compound isdesired, a long wavelength emitter such as a pyrene, bound to thechemiluminescent compound can be used. Alternatively, a fluorescentmolecule can be included in the medium containing the chemiluminescentcompound. In some embodiments, fluorescent molecules will be excited bythe activated chemiluminescent compound and emit at a wavelength longerthan the emission wavelength of the chemiluminescent compound, usuallygreater that 550 nm. It is usually also desirable that the fluorescentmolecules do not absorb at the wavelengths of light used to activate thephotosensitizer. Examples of useful dyes include rhodamine, ethidium,dansyl, Eu(fod)₃, Eu(TTA)₃, Ru(bpy)₃ ⁺⁺ (wherein bpy=2,2′-dipyridyl,etc. In general, these dyes act as acceptors in energy transferprocesses and in some embodiments, have high fluorescent quantum yieldsand do not react rapidly with singlet oxygen. They can be incorporatedinto particles simultaneously with the incorporation of thechemiluminescent compound into the particles.

In some embodiments, the disclosure provides diffractive opticsdetection technology that can be used with, for example, ingestibledevice technology. In certain embodiments, an ingestible device includesthe diffractive optics technology (e.g., diffractive optics detectionsystem). In certain embodiments, the disclosure provides diffractiveoptics technology (e.g., diffractive optics detection systems) that areused outside the body of subject. As an example, an ingestible devicecan be used to obtain one more samples in the body (e.g., in thegastrointestinal tract) of a subject, and the diffractive opticstechnology can be used to analyze the sample(s). Such analysis can beperformed in vivo (e.g., when the ingestible device contains thediffractive optics).

Diffraction is a phenomenon that occurs due to the wave nature of light.When light hits an edge or passes through a small aperture, it isscattered in different directions. But light waves can interfere to add(constructively) and subtract (destructively) from each other, so thatif light hits a non-random pattern of obstacles, the subsequentconstructive and destructive interference will result in a clear anddistinct diffraction pattern. A specific example is that of adiffraction grating, which is of uniformly spaced lines, typicallyprepared by ruling straight, parallel grooves on a surface. Lightincident on such a surface produces a pattern of evenly spaced spots ofhigh light intensity. This is called Bragg scattering, and the distancebetween spots (or ‘Bragg scattering peaks’) is a unique function of thediffraction pattern and the wavelength of the light source. Diffractiongratings, like focusing optics, can be operated in both transmission andreflection modes.

In general, the light used in the diffractive optics can be of anyappropriate wavelength. Exemplary wavelengths include visible light,infrared red (IR) and ultraviolet (UV). Optionally, the light can bemonochromatic or polychromatic. The light can be coherent or incoherent.The light can be collimated or non-collimated. In some embodiments, thelight is coherent and collimated. Generally, any appropriate lightsource may be used, such as, for example, a laser (e.g., a laser diode)or a light emitting diode. In some embodiments, the light source is alaser diode operating at 670 nm wavelength, e.g., at 3 mWatts power.Optionally, an operating wavelength of a laser diode can be 780 nm,e.g., when larger grating periods are used. In certain embodiments, thelight source is a laser, such as, for example, a He—Ne laser, a Nd:YVO4laser, or an argon-ion laser. In some embodiments, the light source is alow power, continuous waver laser.

The diffracted light can be detected using any appropriate lightdetector(s). Examples of light detectors include photodetectors, suchas, for example, position sensitive photodiodes, photomultiplier tubes(PMTs), photodiodes (PDs), avalanche photodiodes (APDs), charged-coupleddevice (CCD) arrays, and CMOS detectors. In some embodiments, thediffracted light is detected via one or more individual photodiodes.

In general, the diffraction grating is made of a material that istransparent in the wavelength of the radiation used to illuminate thesensor. Any appropriate material may be used for the diffraction gratingsubstrate, such as glass or a polymer. Exemplary polymers includepolystyrene polymers (PSEs), cyclo-olefin polymers (COPs), polycarbonatepolymers, polymethyl methacrylates, and methyl methacrylate styrenecopolymers. Exemplary COPs include Zeonex (e.g., Zeonex E48R, ZeonexF52R).

The light may be incident on the diffraction grating any appropriateangle. In some embodiments, the light is incident on the diffractiongrating with an angle of incidence of from 30° to 80° (e.g., from 40° to80°, from 50° to 70°, from 55° to 65°, 60°). Optionally, the system isconfigured so that that diffractive grating and light source can moverelative to each other

In general, the light detector can be positioned with respect to thediffractive grating so that the diffraction grating can be illuminatedat a desired angle of incidence and/or so that diffracted light can bedetected at a desired angle and/or so that diffracted light of a desiredorder can be detected.

The period P of the diffraction grating can be selected as desired. Insome embodiments, the period P is from 0.5 microns to 50 microns (e.g.,from one micron to 15 microns, from one micron to five microns). In someembodiments, the grating is a repeating patter of 1.5 micron and 4.5micron lines with a period of 15 microns.

The height h of the diffraction grating can be selected as desired. Incertain embodiments, the height h is from one nanometer to about 1000nanometers (e.g., from about five nanometers to about 250 nanometers,from five nanometers to 100 nanometers).

In general, the diffractive optics can be prepared using any appropriatemethod, such as, for example, surface ablation, photolithograph (e.g.,UV photolithography), laser etching, electron beam etching, nano-imprintmolding, or microcontact printing.

Optionally, the diffractive optics system can include one or moreadditional optical elements, such as, for example, one or more mirrors,filters and/or lenses. Such optical elements can, for example, bearranged between the light source and the diffractive grating and/orbetween the diffractive grating and the detector.

In some of the embodiments of the devices described herein, a primarybinding partner specifically binds to a secondary binding partnerthrough non-covalent interactions (e.g., electrostatic, van der Waals,hydrophobic effect). In some embodiments, a primary binding partnerspecifically binds to a secondary binding partner via a covalent bond(e.g., a polar covalent bond or a non-polar covalent bond). In someembodiments of any of the devices described herein, the primary and thesecondary binding partner can be interchanged. For example, the primarybinding partner can be biotin, or a derivative thereof, and thesecondary binding partner is avidin, or a derivative thereof. In otherexamples, the primary binding partner can be avidin, or a derivativethereof, and the secondary binding partner is biotin.

In some embodiments, the binding of the primary and the secondarybinding partner is essentially irreversible. In some embodiments, thebinding of the primary and the secondary binding partner is reversible.In some embodiments, the primary binding partner is CaptAvidin™biotin-binding protein and the secondary binding partner is biotin, orvice versa. In some embodiments, the primary binding partner is DSB-X™biotin and the secondary binding partner is avidin, or vice versa. Insome embodiments, the primary binding partner is desthiobiotin and thesecondary binding partner is avidin, or vice versa (Hirsch et al., AnalBiochem. 308(2):343-357, 2002). In some embodiments, the primary bindingpartner is glutathione (GSH) or a derivative thereof, and the secondarybinding partner is glutathione-S-transferase (GST).

In some embodiments, the primary binding partner can bind to a targetanalyte that is a nucleic acid (e.g., a DNA molecule, a RNA molecule).In some embodiments, the primary binding partner comprises a portion ofa nucleic acid that is complementary to the nucleic acid sequence of thetarget analyte.

In some embodiments of any of the devices described herein, the devicecan include a label that binds to the target analyte and does notprevent binding of the target analyte to the primary binding partner. Insome embodiments, the label can amplify the diffraction signal of thetarget analyte.

In some embodiments, the label is from about 1 nm to 200 nm (e.g., about50 nm to about 200 nm).

In some embodiments, the label (e.g., any of the labels describedherein) includes one or more antibodies (e.g., any of the antibodiesand/or antibody fragments described herein). In some embodiments, thelabel is a nanoparticle (e.g., a gold nanoparticle) that includes theprimary binding partner that has a nucleic acid sequence that iscomplementary to the target analyte, and is covalently linked to thenanoparticle.

One or more additional steps can be performed in any of the methodsdescribed herein. In some embodiments, the one or more additional stepsare performed: prior to the binding of the primary binding partner tothe secondary binding partner, after the binding of the primary bindingpartner to the secondary binding partner, prior to the binding of theprimary binding partner to the target analyte, or after the binding ofthe primary binding partner to the target analyte.

In some embodiments of any of the methods described herein, thedetermining step (during which the primary binding partner binds to thetarget analyte is detected) can occur in at least 15 seconds. In someembodiments, the binding of the primary binding partner to the targetanalyte can occur during a period of time of, for example, five at leastseconds.

In some embodiments, the one or more additional steps can include: ablocking of the sensors step, at least one wash step, a capturing step,and/or a filtering step. In some embodiments, the blocking step caninclude blocking a sensor within the ingestible device with a solutioncomprising at least 1% bovine serum albumin (BSA) in a buffered solution(e.g., phosphate buffered saline (PBS), Tris buffered saline (TBS)). Insome embodiments, the at least one wash step can include washing with abuffered solution (e.g., phosphate buffered saline (PBS), Tris bufferedsaline (TBS)). In general, blocking is performed during capsulemanufacture, rather than in vivo.

In some embodiments, the capturing step includes enriching the targetanalyte. In some embodiments, the capturing step includes physicallyseparating the target analyte from the remaining sample using a filter,a pore, or a magnetic bead. In some embodiments, the target analyte iscaptured by size exclusion.

In some embodiments, the disclosure provides methods of obtaining,culturing, and/or detecting target cells and/or target analytes in vivowithin the gastrointestinal (GI) tract or reproductive tract of asubject. Associated devices are also disclosed. The methods and devicesdescribed provide a number of advantages for obtaining and/or analyzingfluid samples from a subject. In some embodiments, diluting the fluidsample increases the dynamic range of analyte detection and/or reducesbackground signals or interference within the sample. For example,interference may be caused by the presence of non-target analytes ornon-specific binding of a dye or label within the sample. In someembodiments, culturing the sample increases the concentration of targetcells and/or target analytes produced by the target cells therebyfacilitating their detection and/or characterization.

In certain embodiments, the methods and devices a described herein maybe used to obtain information regarding bacteria populations in the GItract of a subject. This has a number of advantages and is less invasivethan surgical procedures such as intubation or endoscopy to obtain fluidsamples from the GI tract. The use of an ingestible device as describedherein also allows for fluid samples to be obtained and data to begenerated on bacterial populations from specific regions of the GItract.

In some embodiments, the methods and devices described herein may beused to generate data such as by analyzing the fluid sample, dilutionsthereof or cultured samples for one or more target cells and/or targetanalytes. The data may include, but is not limited to, the types ofbacteria present in the fluid sample or the concentration of bacteria inspecific regions of the GI tract. Such data may be used to determinewhether a subject has an infection, such as Small Intestinal BacterialOvergrowth (SIBO), or to characterize bacterial populations within theGI tract for diagnostic or other purposes. Thus, in some embodiments,analytes disclosed herein are indicative of disorders of thegastrointestinal tract associated with anomalous bacterial populations.

For example, in one aspect, the data may include, but is not limited to,the concentration of bacteria in a specific region of the GI tract thatis one or more of the duodenum, jejunum, ileum, ascending colon,transverse colon or descending colon. In one aspect, the specific regionof the GI tract is the duodenum. In one aspect, the specific region ofthe GI tract is the jejunum. In one aspect, the specific region of theGI tract is the ileum. In one aspect, the specific region of the GItract is the ascending colon. In one aspect, the specific region of theGI tract is the transverse colon. In one aspect, the specific region ofthe GI tract is the descending colon. In a related embodiment, the datamay be generated every one or more days to monitor disease flare-ups, orresponse to the therapeutic agents disclosed herein.

Data may be generated after the device has exited the subject, or thedata may be generated in vivo and stored on the device and recovered exvivo. Alternatively, the data can be transmitted wirelessly from thedevice while the device is passing through the GI tract of the subjector in place within the reproductive tract of the subject.

In some embodiments, a method comprises: providing a device comprisingone or more dilution chambers and dilution fluid; transferring all orpart of a fluid sample obtained from the GI tract or reproductive tractof the subject into the one or more dilution chambers in vivo; andcombining the fluid sample and the dilution fluid to produce one or morediluted samples in the one or more dilution chambers.

In certain embodiments, a method comprises: providing an ingestibledevice comprising one or more dilution chambers; transferring all orpart of a fluid sample obtained from the GI tract into the one or moredilution chambers comprising sterile media; culturing the sample in vivowithin the one or more dilution chambers to produce one or more culturedsamples; and detecting bacteria in the one or more cultured samples.

In some embodiments, a method comprises: providing a device comprisingone or more dilution chambers; transferring all or part of a fluidsample obtained from the GI tract or reproductive tract into the one ormore dilution chambers; combining all or part of the fluid sample with adilution fluid in the one or more dilution chambers; and detecting thetarget analyte in the one or more diluted samples.

In certain embodiments, a device comprises: one or more dilutionchambers for diluting a fluid sample obtained from the GI tract orreproductive tract; and dilution fluid for diluting the sample withinthe one or more dilution chambers.

In some embodiments, the device comprises: one or more dilution chambersfor culturing a fluid sample obtained from the GI tract; sterile mediafor culturing the sample within the one or more dilution chambers; and adetection system for detecting bacteria.

In certain embodiments, a device comprises: one or more dilutionchambers for culturing a fluid sample obtained from the GI tract;sterile media for culturing the sample within the one or more dilutionchambers; and a detection system for detecting bacteria. Also providedis the use of a device as described herein for diluting one or moresamples obtained from the GI tract or reproductive tract of a subject.In one embodiment, there is provided the use of an ingestible device asdescribed herein for detecting target cells and/or target analytes invivo within the gastrointestinal (GI) tract of a subject.

Further provided is a system comprising a device as described herein anda base station. In one embodiment, the device transmits data to the basestation, such as data indicative of the concentration and/or types ofbacteria in the GI tract of the subject. In one embodiment, the devicereceives operating parameters from the base station. Some embodimentsdescribed herein provide an ingestible device for obtaining one or moresamples from the GI tract or reproductive tract of a subject anddiluting and/or culturing all or part of the one or more samples. Theingestible device includes a cylindrical rotatable element having a porton the wall of the cylindrical rotatable element. The ingestible devicefurther includes a shell element wrapping around the cylindricalrotatable element to form a first dilution chamber between thecylindrical rotatable element and the shell element. The shell elementhas an aperture that exposes a portion of the wall of the cylindricalrotatable element to an exterior of the ingestible device.

In certain embodiments, the medical device comprises one or moredilution chambers for receiving a fluid sample from the GI tract orreproductive tract of a subject or a dilution thereof. In someembodiments, one or more dilutions of the fluid sample are cultured inone or more dilution chambers. In certain embodiments, the dilutionchambers each define a known volume, optionally the same volume ordifferent volumes. In some embodiments, the dilution chambers define afluid volume ranging from about 10 μL to about 1 mL. The dilutionchambers may define a fluid volume less than or equal to about 500 μL,less than or equal to about 250 μL, less than or equal to about 100 μL,or less than or equal to about 50 μL. In certain embodiments, thedilution chambers define a fluid volume of greater than or equal toabout 10 μL, greater than or equal to about 20 μL, greater than or equalto about 30 μL, or greater than or equal to about 50 μL. In someembodiments, the dilution chambers define a fluid volume between about10 μL and 500 μL, between about 20 μL and 250 μL, between about 30 μLand 100 μL or about 50 μL.

In some embodiments, dilution fluid in the device is combined with allor part of the fluid sample, or dilution thereof, to produce one or moredilutions. In certain embodiments, the dilution fluid is sterile mediasuitable for culturing one or more target cells within the dilutionchambers.

In certain embodiments, the one or more dilution chambers may be filledwith the dilution fluid prior to a patient ingesting the ingestibledevice. In some embodiments, the dilution fluid may be added into theone or more dilution chambers in vivo from a reservoir of the ingestibledevice. Sampling and dilution of the GI fluid sample may take place invivo. For example, an actuator of the ingestible device may pump thedilution fluid from the reservoir into a dilution chamber when it isdetermined that the ingestible device is located at a predeterminedlocation within the GI tract. In some embodiments, the dilution chamberseach contain a volume of sterile media suitable for culturing a fluidsample from the GI tract or reproductive tract. In certain embodiments,the dilution chambers are at least 95%, at least 97%, at least 98%, orat least 99% full of sterile media. In some embodiments, the dilutionchambers each contain oxygen to facilitate aerobic bacteria growth. Incertain embodiments, a non-dilution chamber comprises oxygen and isadded to one or more of the dilution chambers to facilitate aerobicbacteria growth.

In some embodiments, the culturing may take place in vivo immediatelyafter the GI fluid sample has been diluted. Or alternatively, theculturing may take place ex vivo, e.g., when the ingestible device hasbeen evacuated and recovered such that the dilution chamber containingthe diluted GI fluid sample may be extracted and the culturing may beperformed in a laboratory. The recovery of the ingestible device may beperformed in a similar manner as embodiments described in U.S.Provisional Application No. 62/434,188, filed on Dec. 14, 2016, which isherein expressly incorporated by reference in its entirety.

As used herein “culturing” refers to maintaining target cells in anenvironment that allows a population of one or more target cells toincrease in number through cell division. For example, in someembodiments, “culturing” may include combining the cells with media inan dilution chamber at a temperature that permits cell growth,optionally a temperature found in vivo within the GI tract orreproductive tract of a subject. In certain embodiments, the cells arecultured at a temperature between about 35° C. and 42° C.

As used herein “dilution fluid” refers to a fluid within the device fordiluting a fluid sample from the GI tract or reproductive tract. In someembodiments, the dilution fluid is an aqueous solution. In certainembodiments, the dilution fluid comprises one or more agents thatpromote or inhibit the growth of an organism, such as a fungus orbacteria. In some embodiments, the dilution fluid comprises one or moreagents that facilitate the detection of a target analyte, such as dyesor binding agents for target analytes.

In some embodiments, the dilution fluid is a sterile media. As usedherein, “sterile media” refers to media that does not contain any viablebacteria or other cells that would grow and increase in number throughcell division. Media may be rendered sterile by various techniques knownin the art such as, but not limited to, autoclaving and/or preparing themedia using asceptic techniques. In certain embodiments, the media is aliquid media. Examples of media suitable for culturing bacteria includenutrient broth, Lysogeny Broth (LB) (also known as Luria Broth), Wilkinschalgren, and Tryptic Soy Broth (TSB), Other growth or culture mediaknown in the art may also be used in the methods and devices describedherein. In some embodiments, the media has a carbon source, such asglucose or glycerol, a nitrogen source such as ammonium salts ornitrates or amino acids, as well as salts and/or trace elements andvitamins required for microbial growth. In certain embodiments, themedia is suitable for maintaining eukaryotic cells. In some embodiments,the media comprises one or more agents that promote or inhibit thegrowth of bacteria, optionally agents that promote or inhibit the growthof specific types of bacteria.

In certain embodiments, the media is a selective media. As used herein,“selective media” refers to a media that allows certain types of targetcells to grow and inhibits the growth of other organisms. Accordingly,the growth of cells in a selective media indicates the presence ofcertain types of cells within the cultured sample. For example, in someembodiments, the media is selective for gram-positive or gram-negativebacteria. In certain embodiments, the media contains crystal violet andbile salts (such as found in MacConkey agar) that inhibit the growth ofgram-positive organisms and allows for the selection and isolation ofgram-negative bacteria. In some embodiments, the media contains a highconcentration of salt (NaCl) (such as found in Mannitol salt agar) andis selective for Gram-positive bacteria. In some embodiments, the mediaselectively kills eukaryotic cells or only grows prokaryotic cells, forexample, using a media comprising Triton™ X-100. In certain embodiments,the media selectively kills prokaryotic cells (or alternatively onlygrows eukaryotic cells), for example, using a media that comprisesantibiotics.

In some embodiments, the media is an indicator media. As used herein,“indicator media” refers to a media that contains specific nutrients orindicators (such as, but not limited to neutral red, phenol red, eosiny, or methylene blue) that produce a detectable signal when a certaintype of cells are cultured in the indicator media.

In some embodiments, the disclosure provides a composition comprising adye and optionally a reagent for selective lysis of eukaryotic cells. Incertain embodiments, the composition comprises both a dye and a reagentfor selective lysis of eukaryotic cells. In some embodiments, thecomposition further comprises one or more reagents independentlyselected from the group consisting of: a second reagent for selectivelysis of eukaryotic cells (e.g., Triton X-100), an electrolyte (e.g.,MgCl₂), an anti-fungi reagent (e.g., amphotericin-B), and an antibiotic.In some embodiments, the composition comprises water and is in the formof an aqueous solution. In some embodiments, the composition is a solidor semi-solid. In some embodiments, the compositions described here aresuitable for use in a kit or device for detecting or quantifying viablebacterial cells in a sample. In some embodiments, such a device is aningestible device for detecting or quantifying viable bacterial cells invivo (e.g., in the GI tract). In some embodiments, viable bacterialcells in a sample are detected or quantified in the presence of one ormore antibiotics to determine antibiotic resistance of the bacteria inthe sample. In some embodiments, anomalous bacterial populations in asample may be detected or quantified, for example through the use of onea composition comprising a dye as disclosed herein, to determine whethera subject has an infection, such as Small Intestinal BacterialOvergrowth (SIBO), or to characterize bacterial populations within theGI tract for diagnostic or other purposes.

In some embodiments, a method comprises: (a) contacting the sample witha composition as described herein; and (b) measuring total fluorescenceor rate of change of fluorescence as a function of time of said sample,thereby detecting viable bacterial cells in said sample. In someembodiments, a control as described herein may be employed in themethod. In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample is measuredover multiple time points for an extended period of time in step (b),thereby detecting viable bacterial cells in said sample. In someembodiments, the method further comprises correlating the totalfluorescence or the rate of change of fluorescence as a function of timedetermined in step (b) to the number of viable bacterial cells in thesample. In some embodiments, the rate of change of fluorescence as afunction of time of the sample measured over multiple time points isdetermined and compared to the rate of change of fluorescence as afunction of time of a control measured over the same time points todetermine the number of viable bacterial cells in the sample. In someembodiments, the method does not require ex vivo plating or culturing.In some embodiments, the method does not require aspiration. In someembodiments, the method is performed in vivo (e.g., in an ingestibledevice in vivo). In some embodiments, the method comprises communicatingthe results of the onboard assay(s) to an ex vivo receiver.

In certain embodiments, a kit comprises a composition as describedherein and instructions, e.g., for detecting or quantifying viablebacterial cells in a sample. In some embodiments, a device comprises acomposition as described herein, e.g., for detecting or quantifyingviable bacterial cells in a sample. The detection of live cells, asopposed to the detection of bacterial components (such as endotoxins)which can be present in the sample environment and lead to conflictingresults, is the gold standard of viable plate counting and representsone of the advantages of the compositions and methods described herein.

The systems employ methods, compositions and detection systems found toaccurately and reliably correlate fluorescence to total bacteria count(TBC) in an autonomous, ingestible device, or other similarly-sizeddevice. The compositions include novel combinations of dyes, buffers anddetergents that allow for the selective staining of viable bacterialcells in samples that comprise non-bacterial cells and other componentsthat otherwise make detecting or quantifying live bacterial cellschallenging. In some embodiments, the systems allow for bacteria to bequantified in near real-time and the results to be shared telemetricallyoutside of the device.

In certain embodiments, the disclosure provides a method of assessing ormonitoring the need to treat a subject suffering from or at risk ofovergrowth of bacterial cells in the gastrointestinal tract, whichcomprises: (a) obtaining a sample from the gastrointestinal tract ofsaid subject; (b) contacting the sample with a composition as describedherein; (c) measuring total fluorescence or rate of change offluorescence as a function of time of said sample; and (d) correlatingthe total fluorescence or the rate of change of fluorescence as afunction of time measured in step (c) to the number of viable bacterialcells in the sample, wherein the number of the viable bacterial cellsdetermined in step (e) greater than about 105 CFU/mL indicates a needfor treatment, e.g., with an antibiotic agent as described herein. Insome embodiments, a control as described herein may be employed in themethod. In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample is measuredover multiple time points for an extended period of time in step (c). Insome embodiments, the rate of change of fluorescence as a function oftime of the sample measured over multiple time points is determined andcompared to the rate of change of fluorescence as a function of time ofa control measured over the same time points to determine the number ofviable bacterial cells in the sample. In some embodiments, the methoddoes not require ex vivo plating or culturing. In some embodiments, themethod does not require aspiration. In some embodiments, the method isperformed in vivo (e.g., in an ingestible device in vivo). In someembodiments, the method comprises communicating the results of theonboard assay(s) to an ex vivo receiver. In some embodiments, the methodmay be further used to monitor the subject after the treatment (e.g.,with an antibiotic). In some embodiments, the method may be used toassess the efficacy of the treatment. For example, efficacious treatmentmay be indicated by the decrease of the number of viable bacterial cellsin a sample from the GI tract of the subject post-treatment. Efficacy ofthe treatment may be evaluated by the rate of decrease of the number ofviable bacterial cells in a sample from the GI tract of the subjectpost-treatment. In some embodiments, the method may be used to detectinfection with antibiotic-resistant strains of bacteria in a subject.For instance, such infection may be indicated where the number of viablebacterial cells in a sample from the GI tract of the subject does notsubstantially decrease after antibiotic treatment.

In some embodiments, the disclosure provides an absorbable material,(e.g., absorbable sponge), having absorbed therein a composition asdescribed herein. In some embodiments, the absorbable sponge is AhlstromGrade 6613H (Lot 150191) or Porex PSU-567, having absorbed therein acomposition as described herein. In some embodiments, the absorbablesponge may be prepared by injecting into the absorbable sponge anaqueous solution comprising a composition as described herein, andoptionally further comprising a step of drying the resulting absorbablesponge.

In certain embodiments, the disclosure provides a method for detectingthe presence of viable bacterial cells in a sample, which comprises: (a)fully or partially saturating an absorbable sponge as described herein,or an absorbable sponge prepared as described herein, with the sample;and (b) measuring total fluorescence or rate of change of fluorescenceas a function of time of the fully or partially saturated spongeprepared in step (a), thereby detecting viable bacterial cells. In someembodiments, a control as described herein may be employed in themethod. In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the fully or partiallysaturated sponge is measured over multiple time points for an extendedperiod of time in step (b), thereby detecting viable bacterial cells insaid sample. In some embodiments, the method further comprisescorrelating the total fluorescence or the rate of change of fluorescenceas a function of time measured in step (b) to the number of viablebacterial cells in the sample. In some embodiments, the rate of changeof fluorescence as a function of time of the fully or partiallysaturated sponge measured over multiple time points is determined andcompared to the rate of change of fluorescence as a function of time ofa control measured over the same time points to determine the number ofviable bacterial cells in the sample. In some embodiments, the methoddoes not require ex vivo plating or culturing. In some embodiments, themethod does not require aspiration. In some embodiments, the method isperformed in vivo (e.g., in an ingestible device in vivo). In someembodiments, the method comprises communicating the results of theonboard assay(s) to an ex vivo receiver.

In one aspect, provided herein is a kit comprising an absorbable spongeas described herein and instructions, e.g., for detecting or quantifyingviable bacterial cells in a sample. In another aspect, provided hereinis a device comprising an absorbable sponge as described herein, e.g.,for detecting or quantifying viable bacterial cells in a sample.

In certain embodiments, the disclosure provides a method of assessing ormonitoring the need to treat a subject suffering from or at risk ofovergrowth of bacterial cells in the gastrointestinal tract, whichcomprises: (a) obtaining a sample from the gastrointestinal tract ofsaid subject; (b) fully or partially saturating an absorbable spongedescribed herein, or an absorbable sponge prepared as described herein,with the sample; (c) measuring total fluorescence or rate of change offluorescence as a function of time of the fully or partially saturatedsponge prepared in step (b); (d) correlating the total fluorescence orthe rate of change of fluorescence as a function of time measured instep (c) to the number of viable bacterial cells in the sample, whereinthe number of the viable bacterial cells as determined in step (e)greater than about 10⁵ CFU/mL indicates a need for treatment, e.g., withan antibiotic agent as described herein. In some embodiments, a controlas described herein may be employed in the method. In some embodiments,the total fluorescence or the rate of change of fluorescence as afunction of time of the fully or partially saturated sponge is measuredover multiple time points for an extended period of time in step (c). Insome embodiments, the rate of change of fluorescence as a function oftime of the fully or partially saturated sponge measured over multipletime points is determined and compared to the rate of change offluorescence as a function of time of a control measured over the sametime points to determine the number of viable bacterial cells in thesample. In some embodiments, the method does not require ex vivo platingor culturing. In some embodiments, the method does not requireaspiration. In some embodiments, the method is performed in vivo (e.g.,in an ingestible device in vivo). In some embodiments, the methodcomprises communicating the results of the onboard assay(s) to an exvivo receiver. In some embodiments, the method may be further used tomonitor the subject after the treatment (e.g., with an antibiotic). Insome embodiments, the method may be used to assess the efficacy of thetreatment. For example, efficacious treatment may be indicated by thedecrease of the number of viable bacterial cells in a sample from the GItract of the subject post-treatment. Efficacy of the treatment may beevaluated by the rate of decrease of the number of viable bacterialcells in a sample from the GI tract of the subject post-treatment. Insome embodiments, the method may be used to detect infection withantibiotic-resistant strains of bacteria in a subject. For instance,such infection may be indicated where the number of viable bacterialcells in a sample from the GI tract of the subject does notsubstantially decrease after antibiotic treatment

In certain embodiments, the disclosure provides and ingestible devicecomprising a housing; a first opening in the wall of the housing; asecond opening in the first end of the housing; and a chamber connectingthe first opening and the second opening, wherein at least a portion ofthe chamber forms a sampling chamber within the ingestible device. Insome embodiments, the sampling chamber is configured to hold anabsorbable sponge described herein. In some embodiments, the samplingchamber is configured to hold a sample obtained from a gastrointestinal(GI) tract of a body. In some embodiments, the ingestible device isindividually calibrated (for example, by comparing to a positive ornegative control as described herein), wherein the fluorescentproperties of the absorbable sponge held in the sampling chamber of thedevice are determined prior to the introduction of the sample. Theingestible device as described herein is useful for detecting orquantifying viable bacterial cells in vivo. In some embodiments,provided herein is a method for detecting or quantifying viablebacterial cells in a GI tract sample in vivo using an ingestible deviceas described herein. In some embodiments, provided herein is a method ofassessing or monitoring the need to treat a subject suffering from or atrisk of overgrowth of bacterial cells in the GI tract in vivo using aningestible device as described herein. In some embodiments, providedherein is a method of altering the treatment regimen of a subjectsuffering from or at risk of overgrowth of bacterial cells in the GItract in vivo using an ingestible device as described herein. In oneaspect, the subject is a subject suffering from or at risk of overgrowthof bacterial cells in the duodenum. In one aspect, the subject is asubject suffering from or at risk of overgrowth of bacterial cells inthe jejunum. In one aspect, the subject is a subject suffering from orat risk of overgrowth of bacterial cells in the ileum. In one aspect,the subject is a subject suffering from or at risk of overgrowth ofbacterial cells in the ascending colon. In one aspect, the subject is asubject suffering from or at risk of overgrowth of bacterial cells inthe transverse colon. In one aspect, the subject is a subject sufferingfrom or at risk of overgrowth of bacterial cells in the descendingcolon. In some embodiments, the method may be further used to monitorthe subject after the treatment (e.g., with an antibiotic). In someembodiments, the method may be used to assess the efficacy of thetreatment. For example, efficacious treatment may be indicated by thedecrease of the number of viable bacterial cells in a sample from the GItract of the subject post-treatment. Efficacy of the treatment may beevaluated by the rate of decrease of the number of viable bacterialcells in a sample from the GI tract of the subject post-treatment. Insome embodiments, the method may be used to detect infection withantibiotic-resistant strains of bacteria in a subject. For instance,such infection may be indicated where the number of viable bacterialcells in a sample from the GI tract of the subject does notsubstantially decrease after antibiotic treatment. In some embodiments,the method is performed autonomously and does not require instructions,triggers or other inputs from outside the body after the device has beeningested.

“Eukaryotic” as recited herein relates to any type of eukaryoticorganism excluding fungi, such as animals, in particular animalscontaining blood, and comprises invertebrate animals such as crustaceansand vertebrates. Vertebrates comprise both cold-blooded (fish, reptiles,amphibians) and warm blooded animal (birds and mammals). Mammalscomprise in particular primates and more particularly humans

“Selective lysis” as used herein is obtained in a sample when thepercentage of bacterial cells in that sample that remain intact issignificantly higher (e.g. 2, 5, 10, 20, 50, 100, 250, 500, or 1,000times more) than the percentage of the eukaryotic cells in that samplethat remain intact, upon treatment of or contact with a composition ordevice as described herein.

In some embodiments, the dye suitable for use herein is a dye that iscapable of being internalized by a viable cell, binding to or reactingwith a target component of the viable cell, and having fluorescenceproperties that are measurably altered when the dye is bound to orreacted with the target component of the viable cell. In someembodiments, the dye herein is actively internalized by penetratingviable cells through a process other than passible diffusion across cellmembranes. Such internalization includes, but is not limited to,internalization through cell receptors on cell surfaces or throughchannels in cell membranes. In some embodiments, the target component ofa viable cell to which the dye is bound to or reacted with is selectedfrom the group consisting of: nucleic acids, actin, tubulin, enzymes,nucleotide-binding proteins, ion-transport proteins, mitochondria,cytoplasmic components, and membrane components. In some embodiments,the dye suitable for use herein is a fluorogenic dye that is capable ofbeing internalized and metabolized by a viable cell, and wherein saiddye fluoresces when metabolized by the viable cell. In some embodiments,the dye is a chemiluminescent dye that is capable of being internalizedand metabolized by a viable cell, and wherein said dye becomeschemiluminescent when metabolized by the viable cell.

In some embodiments, the composition comprises a dye that fluoresceswhen bond to nucleic acids. Examples of such dyes include, but are notlimited to, acridine orange (U.S. Pat. No. 4,190,328); calcein-AM (U.S.Pat. No. 5,314,805); DAPI; Hoechst 33342; Hoechst 33258; PicoGreen™;SYTO® 16; SYBR® Green I; Texas Red®; Redmond Red™; Bodipy® Dyes; OregonGreen™; ethidium bromide; and propidium iodide.

In some embodiments, the composition comprises a lipophilic dye thatfluoresces when metabolized by a cell. In some embodiments, the dyefluoresces when reduced by a cell or a cell component. Examples of dyesthat fluoresce when reduced include, but are not limited to, resazurin;C¹²-resazurin; 7-hydroxy-9H-(1,3dichloro-9,9-dimethylacridin-2-ol)N-oxide;6-chloro-9-nitro-5-oxo-SH-benzo[a]phenoxazine; and tetrazolium salts. Insome embodiment, the dye fluoresces when oxidized by a cell or a cellcomponent. Examples of such dyes include, but are not limited to,dihydrocalcein AM; dihydrorhodamine 123; dihydroethidium;2,3,4,5,6-pentafluorotetramethyldihydrorosamine; and 3′-(p-aminophenyl)fluorescein.

In some embodiments, the composition comprises a dye that becomeschemiluminescent when oxidized by a cell or a cell component, such asluminol.

In some embodiments, the composition comprises a dye that fluoresceswhen de-acetylated and/or oxidized by a cell or a cell component.Examples of such dyes include, but are not limited to,dihydrorhodamines; dihydrofluoresceins; 2′,7′-dichlorodihydrofluoresceindiacetate; 5-(and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate;and chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester.

In some embodiments, the composition comprises a dye that fluoresceswhen reacted with a peptidase. Examples of such dyes include, but arenot limited to, (CBZ-Ala-Ala-Ala-Ala)2-R110 elastase 2;(CBZ-Ala-Ala-Asp)2-R110 granzyme B; and 7-amino-4-methylcoumarin,N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide.

In some embodiments, the composition comprises a dye selected from thegroup consisting of resazurin, FDA, Calcein AM, and SYTO® 9. In someembodiments, the dye is FDA or SYTO® 9.

SYTO® 9, when used alone, labels the nucleic acid of bacteria cells. Theexcitation/emission wavelengths for SYTO® 9 is 480/500 nm, with thebackground remaining non-fluorescent. See, e.g., J. Appl. Bacteriol. 72,410 (1992); Lett. Appl. Microbiol. 13, 58 (1991); Curr. Microbiol. 4,321 (1980); J. Microbiol. Methods 13, 87 (1991); and Microbiol. Rev. 51,365 (1987); and J. Med. Microbiol. 39, 147 (1993).

FDA is a non-polar, non-fluorescent compound that can cross themembranes of mammalian and bacterial cells. The acetyl esterases(present only within viable cells) hydrolyze the FDA into thefluorescent compound fluorescein. Fluorescein is a fluorescent polarcompound that is retained within these cells. Living cells can bevisualized in a photospectrometer when assayed with an excitationwavelength of 494 nm and an emission wavelength of 518 nm. See, e.g.,Brunius, G. (1980). Technical aspects of the use of 3′,6′-Diacetylfluorescein for vital fluorescent staining of bacteria. CurrentMicrobiol. 4: 321-323; Jones, K. H. and Senft, J. A. (1985). An improvedmethod to determine cellviability by simultaneous staining withfluorescein diacetate-propidium iodide. J. Histochem. Cytochem. 33:77-79; Ross, R. D., Joneckis, C. C., Ordonez, J. V., Sisk, A. M., Wu, R.K., Hamburger, A. W., and Nora, R. E. (1989). Estimation of cellsurvival by flow cytometric quantification of fluoresceindiacetate/propidium iodide viable cell number. Cancer Research. 49:3776-3782.

Calcein-AM, which is an acetoxylmethyl ester of calcein, is highlylipophilic and cell permeable. Calcein-AM in itself is not fluorescent,but the calcein generated by esterase in a viable cell emits a greenfluorescence with an excitation wavelength of 490 nm and an emission of515 nm. Therefore, Calcein-AM can only stain viable cells. See, e.g.,Kimura, K., et al., Neurosci. Lett., 208, 53 (1998); Shimokawa, I., etal., J. Geronto., 51a, b49 (1998); Yoshida, S., et al., Clin. Nephrol.,49, 273 (1998); and Tominaga, H., et al., Anal. Commun., 36, 47 (1999).

Resazuirn (also known as Alamar Blue) is a blue compound that can bereduced to pink resorufin which is fluorescent. This dye is mainly usedin viability assays for mammalian cells. C¹²-resazurin has better cellpermeability than resazurin. When lipohilic C¹²-resazurin crosses thecell membranes, it is subsequently reduced by living cells to make a redfluorescent resorufin. The adsorption/emission of C¹²-resazurin is563/587 nm. See, e.g., Appl Environ Microbiol 56, 3785 (1990); J DairyRes 57, 239 (1990); J Neurosci Methods 70, 195 (1996); J Immunol Methods210, 25 (1997); J Immunol Methods 213, 157 (1998); Antimicrob AgentsChemother 41, 1004 (1997).

In some embodiments, the composition optionally further comprises areagent for selective lysis of eukaryotic cells. In some embodiments,the composition comprises a dye as described herein and a reagent forselective lysis of eukaryotic cells. In some embodiments, the reagentfor selective lysis of eukaryotic cells is a detergent, such as anon-ionic or an ionic detergent. Examples of the reagent for selectivelysis of eukaryotic cells include, but are not limited to,alkylglycosides, Brij 35 (C12E23 Polyoxyethyleneglycol dodecyl ether),Brij 58 (C16E20 Polyoxyethyleneglycol dodecyl ether), Genapol, glucanidssuch as MEGA-8, -9, -10, octylglucoside, Pluronic F127, Triton X-100(C₁₄H₂₂O(C₂H₄O)_(n)), Triton X-114 (C₂₄H₄₂O₆), Tween 20 (Polysorbate 20)and Tween 80 (Polysorbate 80), Nonidet P40, deoxycholate, reduced TritonX-100 and/or Igepal CA 630. In some embodiments, the compositioncomprises a dye as described herein and deoxycholate (e.g., sodiumdeoxycholate) as a reagent for selective lysis of eukaryotic cells. Insome embodiments, the composition comprises deoxycholate at aconcentration selected from 0.0001% to 1 wt %. In some embodiments, thecomposition comprises deoxycholate at a concentration of 0.005 wt %. Insome embodiments, the composition may comprise more than one reagent forselective lysis of eukaryotic cells.

In some embodiments, the composition may comprise two different reagentsfor selective lysis of eukaryotic cells. In some instances, when morethan one selective lysis reagents are used, more effective and/orcomplete selective lysis of eukaryotic cells in a sample may beachieved. For example, the composition may comprise deoxycholate (e.g.,sodium deoxycholate) and Triton X-100 as two different reagents forselective lysis of eukaryotic cells. In some embodiments, thecomposition comprises deoxycholate (e.g., sodium deoxycholate) at aconcentration selected from 0.0001% to 1 wt % (e.g., 0.005 wt %) andTriton X-100 at a concentration selected from 0.1 to 0.05 wt %.

In some embodiments, after a sample (e.g., a biological sample) istreated or contacted with a composition comprising a dye and one or morereagents for selective lysis of eukaryotic cells as described herein,the eukaryotic cells (e.g., animal cells) in the sample are selectivelylysed whereby a substantial percentage (e.g., more than 20%, 40%, 60%,80%, 90% or even more that 95%) of the bacterial cells in the samesample remains intact or alive.

In some embodiments, the composition does not comprise a reagent forselective lysis of eukaryotic cells, and such a composition is usefulfor detecting or quantifying viable bacterial cells in a sample (e.g.,an environmental sample such as a water sample) that does not containany eukaryotic cells.

In some embodiments, the composition further comprises an electrolyte,such as a divalent electrolyte (e.g., MgCl₂). In some embodiments, thecomposition comprises MgCl₂ at a concentration selected from 0.1 mM to100 mM (e.g., a concentration selected from 0.5 mM to 50 mM).

In some embodiments, the composition further comprises water and is in aform of an aqueous solution. In some embodiments, the composition has apH selected from 5-8 (e.g., a pH selected from 6-7.8, such as pH being6.0). In some embodiments, the composition is a solid or a semi-solid.

In some embodiments, the composition further comprises an anti-fungalagent. Suitable anti-fungal agents for use herein include, but are notlimited to, fungicidal and fungistatic agents including terbinafine,itraconazole, micronazole nitrate, thiapendazole, tolnaftate,clotrimazole and griseofulvin. In some embodiments, the anti-fungalagent is a polyene anti-fungal agent, such as amphotericin-B, nystatin,and pimaricin.

In some embodiments, the composition does not contain any anti-fungalagent. In some embodiments, the composition contains broad spectrumantibiotics but not any anti-fungal agent. Such compositions that do notcontain anti-fungal agents but contain broad spectrum antibiotics may beuseful in detecting or quantifying fungi (e.g., yeast) in a sample.

In some embodiments, the composition does not contain any anti-fungalagent, any antibiotics or any anti-mammalian agent. Such compositionsthat do not selectively lyse mammalian cells may be useful in detectingor quantifying mammalian cells (e.g., cells from the GI tract) in asample since many dyes have a higher affinity for mammalian as comparedto bacteria or fungi cells. In some embodiments, the compositioncontains broad spectrum antibiotics and one or more anti-fungal agents.Such compositions that contain anti-fungal agents and broad spectrumantibiotics may be useful in detecting or quantifying mammalian cells(e.g., cells from the GI tract) in a sample. The detection orquantification of mammalian cells may be useful for determining cellturnover in a subject. High cell turnover is sometimes associated with aGI injury (e.g., lesion), the presence of a tumor(s), orradiation-induced colitis or radiation enteropathy.

In some embodiments, the composition further comprises an antibioticagent as described herein. Such a composition may be useful in detectingor quantifying antibiotic-resistant strains of bacteria in a sample.

In certain embodiments, the composition comprises Triton X-100,deoxycholate, resazurin, and MgCl₂. In some embodiments, the compositioncomprises Triton X-100, deoxycholate, resazurin, amphotericin-B andMgCl₂. In some embodiments, the composition comprises 0.1 wt % or 0.05wt % Triton X-100; 0.005 wt % deoxycholate; 10 mM resazurin; 2.5 mg/Lamphotericin-B and 50 mM MgCl₂. In some embodiments, the composition hasa pH of 6.0.

In certain embodiments, the compositions are suitable for use in a kitor device, e.g., for detecting or quantifying viable bacterial cells ina sample. In some embodiments, such a device is an ingestible device fordetecting or quantifying viable bacterial cells in vivo (e.g., in the GItract).

FIG. 62 illustrates a nonlimiting example of a system for collecting,communicating and/or analyzing data about a subject, using an ingestibledevice as disclosed herein. For example, an ingestible device may beconfigured to communicate with an external base station. As an example,an ingestible device can have a communications unit that communicateswith an external base station which itself has a communications unit.FIG. 62 illustrates exemplary implementation of such an ingestibledevice. As shown in FIG. 62, a subject ingests an ingestible device asdisclosed herein. Certain data about the subject (e.g., based on acollected sample) and/or the location of the ingestible device in the GItract of the subject is collected or otherwise available and provided toa mobile device, which then forwards the data via the internet and aserver/data store to a physician's office computer. The informationcollected by the ingestible device is communicated to a receiver, suchas, for example, a watch or other object worn by the subject. Theinformation is then communicated from the receiver to the mobile devicewhich then forwards the data via the internet and a server/data store toa physician's office computer. The physician is then able to analyzesome or all of the data about the subject to provide recommendations,such as, for example, delivery a therapeutic agent. While FIG. 62 showsa particular approach to collecting and transferring data about asubject, the disclosure is not limited. As an example, one or more ofthe receiver, mobile device, internet, and/or server/data store can beexcluded from the data communication channel. For example, a mobiledevice can be used as the receiver of the device data, e.g., by using adongle. In such embodiments, the item worn by the subject need not bepart of the communication chain. As another example, one or more of theitems in the data communication channel can be replaced with analternative item. For example, rather than be provided to a physician'soffice computer, data may be provided to a service provider network,such as a hospital network, an HMO network, or the like. In someembodiments, subject data may be collected and/or stored in one location(e.g., a server/data store) while device data may be collected and/orstored in a different location (e.g., a different server/data store).

Locations of Treatment

In some embodiments, the TLR agonist is delivered at a location in thelarge intestine of the subject. In some embodiments, the location is inthe proximal portion of the large intestine. In some embodiments, thelocation is in the distal portion of the large intestine.

In some embodiments, the TLR agonist is delivered at a location in theascending colon of the subject. In some embodiments, the location is inthe proximal portion of the ascending colon. In some embodiments, thelocation is in the distal portion of the ascending colon.

In some embodiments, the TLR agonist is delivered at a location in thececum of the subject. In some embodiments, the location is in theproximal portion of the cecum. In some embodiments, the location is inthe distal portion of the cecum.

in some embodiments, the TLR agonist is delivered at a location in thesigmoid colon of the subject. In some embodiments, the location is inthe proximal portion of the sigmoid colon. In some embodiments, thelocation is in the distal portion of the sigmoid colon.

In some embodiments, the TLR agonist is delivered at a location in thetransverse colon of the subject. In some embodiments, the location is inthe proximal portion of the transverse colon. In some embodiments, thelocation is in the distal portion of the transverse colon.

In some embodiments, the TLR agonist is delivered at a location in thedescending colon of the subject. In some embodiments, the location is inthe proximal portion of the descending colon. In some embodiments, thelocation is in the distal portion of the descending colon.

In some embodiments, the TLR agonist is delivered at a location in thesmall intestine of the subject. In some embodiments, the location is inthe proximal portion of the small intestine. In some embodiments, thelocation is in the distal portion of the small intestine.

In some embodiments, the TLR agonist is delivered at a location in theduodenum of the subject. In some embodiments, the location is in theproximal portion of the duodenum. In some embodiments, the location isin the distal portion of the duodenum.

In some embodiments, the TLR agonist is delivered at a location in thejejunum of the subject. In some embodiments, the location is in theproximal portion of the jejunum. In some embodiments, the location is inthe distal portion of the jejunum.

In some embodiments, the TLR agonist is delivered at a location in theduodenum of the subject and is not delivered at other locations in thegastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the duodenum of the subject and is notdelivered at other locations in the gastrointestinal tract, wherein asite of disease is in the duodenum and no site of disease is present atother locations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in theduodenum of the subject and is not delivered at other locations in thegastrointestinal tract, wherein a first site of disease is in theduodenum and a second site of disease is in the stomach and no site ofdisease is present at other locations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in theproximal duodenum of the subject and is not delivered at other locationsin the gastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the proximal duodenum of the subject and isnot delivered at other locations in the gastrointestinal tract, whereina site of disease is in the duodenum and no site of disease is presentat other locations in the gastrointestinal tract. In some embodiments,the TLR agonist is delivered at a location in the proximal duodenum ofthe subject and is not delivered at other locations in thegastrointestinal tract, wherein a first site of disease is in theduodenum and a second site of disease is in the stomach and no site ofdisease is present at other locations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in thejejunum of the subject and is not delivered at other locations in thegastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the jejunum of the subject and is notdelivered at other locations in the gastrointestinal tract, wherein asite of disease is in the jejunum and no site of disease is present atother locations in the gastrointestinal tract. In some embodiments, theTLR agonist is delivered at a location in the jejunum of the subject andis not delivered at other locations in the gastrointestinal tract,wherein a first site of disease is in the jejunum and a second site ofdisease is in the ileum and no site of disease is present at otherlocations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in theproximal portion of the jejunum of the subject and is not delivered atother locations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in theproximal portion of the jejunum of the subject and is not delivered atother locations in the gastrointestinal tract, wherein a site of diseaseis in the jejunum and no site of disease is present at other locationsin the gastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the proximal portion of the jejunum of thesubject and is not delivered at other locations in the gastrointestinaltract, wherein a first site of disease is in the jejunum and a secondsite of disease is in the ileum and no site of disease is present atother locations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in thedistal portion of the jejunum of the subject and is not delivered atother locations in the gastrointestinal tract. In some embodiments, theTLR agonist is delivered at a location in the distal portion of thejejunum of the subject and is not delivered at other locations in thegastrointestinal tract, wherein a site of disease is in the jejunum andno site of disease is present at other locations in the gastrointestinaltract. In some embodiments, the TLR agonist is delivered at a locationin the distal portion of the jejunum of the subject and is not deliveredat other locations in the gastrointestinal tract, wherein a first siteof disease is in the jejunum and a second site of disease is in theileum and no site of disease is present at other locations in thegastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in theileum of the subject. In some embodiments, the location is in theproximal portion of the ileum. In some embodiments, the location is inthe distal portion of the ileum.

In some embodiments, the TLR agonist is delivered at a location in theileum of the subject and is not delivered at other locations in thegastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the ileum of the subject and is not deliveredat other locations in the gastrointestinal tract, wherein a site ofdisease is in the ileum and no site of disease is present at otherlocations in the gastrointestinal tract. In some embodiments, the TLRagonist is delivered at a location in the ileum of the subject and isnot delivered at other locations in the gastrointestinal tract, whereina first site of disease is in the ileum and a second site of disease isin the cecum and no site of disease is present at other locations in thegastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the ileum of the subject and is not deliveredat other locations in the gastrointestinal tract, wherein a first siteof disease is in the ileum and a second site of disease is in the cecumand/or ascending colon, and no site of disease is present at otherlocations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in theproximal portion of the ileum of the subject and is not delivered atother locations in the gastrointestinal tract. In some embodiments, theTLR agonist is delivered at a location in the proximal portion of theileum of the subject and is not delivered at other locations in thegastrointestinal tract, wherein a site of disease is in the ileum and nosite of disease is present at other locations in the gastrointestinaltract. In some embodiments, the TLR agonist is delivered at a locationin the proximal portion of the ileum of the subject and is not deliveredat other locations in the gastrointestinal tract, wherein a first siteof disease is in the ileum and a second site of disease is in the cecumand no site of disease is present at other locations in thegastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the proximal portion of the ileum of thesubject and is not delivered at other locations in the gastrointestinaltract, wherein a first site of disease is in the ileum and a second siteof disease is in the cecum and/or ascending colon, and no site ofdisease is present at other locations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in thedistal portion of the ileum of the subject and is not delivered at otherlocations in the gastrointestinal tract. In some embodiments, the TLRagonist is delivered at a location in the distal portion of the ileum ofthe subject and is not delivered at other locations in thegastrointestinal tract, wherein a site of disease is in the ileum and nosite of disease is present at other locations in the gastrointestinaltract. In some embodiments, the TLR agonist is delivered at a locationin the distal portion of the ileum of the subject and is not deliveredat other locations in the gastrointestinal tract, wherein a first siteof disease is in the ileum and a second site of disease is in the cecumand no site of disease is present at other locations in thegastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the distal portion of the ileum of thesubject and is not delivered at other locations in the gastrointestinaltract, wherein a first site of disease is in the ileum and a second siteof disease is in the cecum and/or ascending colon, and no site ofdisease is present at other locations in the gastrointestinal tract.

In some embodiments, the TLR agonist is delivered at a location in thececum of the subject and is not delivered at other locations in thegastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the distal portion of the cecum of thesubject and is not delivered at other locations in the gastrointestinaltract, wherein a site of disease is in the cecum and/or ascending colon,and no site of disease is present at other locations in thegastrointestinal tract. In some embodiments, the TLR agonist isdelivered at a location in the distal portion of the ileum or theproximal portion of the ascending colon of the subject and is notdelivered at other locations in the gastrointestinal tract, wherein afirst site of disease is in the cecum and a second site of disease is inthe ascending colon, and no site of disease is present at otherlocations in the gastrointestinal tract.

In some embodiments, a site of disease is in the colon and the TLRagonist is released in the colon, such as in the cecum. In someembodiments, a site of disease is in the ascending colon and the TLRagonist is released in the ascending colon, such as in the cecum. Insome embodiments, a site of disease is in the ileum and the TLR agonistis released in the ileum.

In some embodiments the subject is diagnosed with ileal Crohn's diseaseand the TLR agonist is released in the ileum.

In some embodiments the subject is diagnosed with ileal colonic Crohn'sdisease and the TLR agonist is released in both the ileum and the colon.In some more particular embodiments, the TLR agonist is released in boththe ileum and the colon from the same ingestble device. In some moreparticular embodiments, the TLR agonist is released in the ileum from afirst ingestble device and in the colon from a second ingestible device,wherein the first ingestble device and the second ingestible device areingested at substantially the same time or at different times.

In some embodiments the subject is diagnosed with colitis throughout thecolon and the TLR agonist is released (a) in the cecum, (b) in the cecumand in the transverse colon, and/or release (c) in the descending colon.

In some embodiments the subject is diagnosed with right sided colitisand the TLR agonist is released in the transverse colon or in thedescending colon.

In some embodiments the subject is diagnosed with rectosigmoidal colitisand the TLR agonist is released in the descending colon.

In some embodiments, the location at which the TLR agonist is deliveredis proximate to a site of disease. The site of disease may be, forexample, an injury, inflamed tissue, or one or more lesions. In someembodiments, the location at which the TLR agonist is delivered isproximate to one or more sites of disease. In some embodiments, the TLRagonist is delivered 150 cm or less from the one or more sites ofdisease. In some embodiments, the TLR agonist is delivered 125 cm orless from the one or more sites of disease. In some embodiments, the TLRagonist is delivered 100 cm or less from the one or more sites ofdisease. In some embodiments, the TLR agonist is delivered 50 cm or lessfrom the one or more sites of disease. In some embodiments, the TLRagonist is delivered 40 cm or less from the one or more sites ofdisease. In some embodiments, the TLR agonist is delivered 30 cm or lessfrom the one or more sites of disease. In some embodiments, the TLRagonist is delivered 20 cm or less from the one or more sites ofdisease. In some embodiments, the TLR agonist is delivered 10 cm or lessfrom the one or more sites of disease. In some embodiments, the TLRagonist is delivered 5 cm or less from the one or more sites of disease.In some embodiments, the TLR agonist is delivered 2 cm or less from theone or more sites of disease. In some embodiments, the method furthercomprises using an ingestible device to deliver the TLR agonist andusing localization methods disclosed herein (e.g., such as discussed inExample 13 below) to determine the location of the ingestible devicewithin the GI tract (e.g., relative to the site of disease). In someembodiments, the method further comprises using an ingestible device todeliver the TLR agonist and determining the period of time since theingestible device was ingested to determine the location of theingestible device within the GI tract (e.g., relative to the site ofdisease). In some embodiments, the method further comprises identifyingthe one or more sites of disease by a method comprising imaging of thegastrointestinal tract. In some embodiments, imaging of thegastrointestinal tract comprises video imaging. In some embodiments,imaging of the gastrointestinal tract comprises thermal imaging. In someembodiments, imaging of the gastrointestinal tract comprises ultrasoundimaging. In some embodiments, imaging of the gastrointestinal tractcomprises Doppler imaging.

In some embodiments the method does not comprise releasing more than 20%of the TLR agonist at a location that is not proximate to a site ofdisease. In some embodiments the method does not comprise releasing morethan 10% of the TLR agonist at a location that is not proximate to asite of disease. In some embodiments the method does not comprisereleasing more than 5% of the TLR agonist at a location that is notproximate to a site of disease. In some embodiments the method does notcomprise releasing more than 4% of the TLR agonist at a location that isnot proximate to a site of disease. In some embodiments the method doesnot comprise releasing more than 3% of the TLR agonist at a locationthat is not proximate to a site of disease. In some embodiments themethod does not comprise releasing more than 2% of the TLR agonist at alocation that is not proximate to a site of disease. In some embodimentsthe method comprises releasing at least 80% of the TLR agonist at alocation proximate to a site of disease. In some embodiments the methodcomprise releasing at least 90% of the TLR agonist at a locationproximate to a site of disease. In some embodiments the method comprisesreleasing at least 95% of the TLR agonist at a location proximate to asite of disease. In some embodiments the method comprises releasing atleast 96% of the TLR agonist at a location proximate to a site ofdisease. In some embodiments the method comprises releasing at least 97%of the TLR agonist at a location proximate to a site of disease. In someembodiments the method comprises releasing at least 98% of the TLRagonist at a location proximate to a site of disease. In someembodiments, the at least 80%, at least 90%, at least 95%, at least 96%,at least 97%, or at least 98% of the TLR agonist is delivered 150 cm orless from the one or more sites of disease. In some embodiments, the atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, or atleast 98% of the TLR agonist is delivered 125 cm or less from the one ormore sites of disease. In some embodiments, the at least 80%, at least90%, at least 95%, at least 96%, at least 97%, or at least 98% of theTLR agonist is delivered 100 cm or less from the one or more sites ofdisease. In some embodiments, the at least 80%, at least 90%, at least95%, at least 96%, at least 97%, or at least 98% of the TLR agonist isdelivered 50 cm or less from the one or more sites of disease. In someembodiments, the at least 80%, at least 90%, at least 95%, at least 96%,at least 97%, or at least 98% of the TLR agonist is delivered 40 cm orless from the one or more sites of disease. In some embodiments, the atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, or atleast 98% of the TLR agonist is delivered 30 cm or less from the one ormore sites of disease. In some embodiments, the at least 80%, at least90%, at least 95%, at least 96%, at least 97%, or at least 98% of theTLR agonist is delivered 20 cm or less from the one or more sites ofdisease. In some embodiments, the at least 80%, at least 90%, at least95%, at least 96%, at least 97%, or at least 98% of the TLR agonist isdelivered 10 cm or less from the one or more sites of disease. In someembodiments, the at least 80%, at least 90%, at least 95%, at least 96%,at least 97%, or at least 98% of the TLR agonist is delivered 5 cm orless from the one or more sites of disease. In some embodiments, the atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, or atleast 98% of the TLR agonist is delivered 2 cm or less from the one ormore sites of disease. In some embodiments, the method further comprisesusing an ingestible device to deliver the TLR agonist and usinglocalization methods disclosed herein (e.g., such as discussed inExample 13 below) to determine the location of the ingestible devicewithin the GI tract (e.g., relative to the site of disease). In someembodiments, the method further comprises using an ingestible device todeliver the TLR agonist and determining the period of time since theingestible device was ingested to determine the location of theingestible device within the GI tract (e.g., relative to the site ofdisease).

In some embodiments, the amount of TLR agonist that is delivered is aHuman Equivalent Dose.

In some embodiments the method comprises releasing the TLR agonist at alocation that is proximate to a site of disease, wherein the TLR agonistand, if applicable, any carriers, excipients or stabilizers admixed withthe TLR agonist, are substantially unchanged, at the time of release ofthe TLR agonist at the location, relatively to the time ofadministration of the composition to the subject.

In some embodiments the method comprises releasing the TLR agonist at alocation that is proximate to a site of disease, wherein the TLR agonistand, if applicable, any carriers, excipients or stabilizers admixed withthe TLR agonist, are substantially unchanged by any physiologicalprocess (such as, but not limited to, degradation in the stomach), atthe time of release of the TLR agonist at the location, relatively tothe time of administration of the composition to the subject.

In some embodiments, the TLR agonist is delivered to the location bymucosal contact.

In some embodiments, a method of treatment disclosed herein includesdetermining the level of TLR agonist at a site of disease or a locationin the gastrointestinal tract of the subject that is proximate to one ormore sites of disease. In some examples, a method of treatment asdescribed herein can include determining the level of TLR agonist at asite of disease or a location in the gastrointestinal tract of thesubject that is proximate to one or more sites of disease within a timeperiod of about 10 minutes to about 10 hours following administration ofthe device.

In some examples, a method of treatment disclosed herein includesdetermining the level of the TLR agonist at a site of disease or alocation in the gastrointestinal tract of the subject that is proximateto one or more sites of disease at a time point following administrationof the device that is elevated as compared to a level of the TLR agonistat the same site of disease or location at substantially the same timepoint in a subject following systemic administration of an equal amountof the TLR agonist.

In some examples where the TLR agonist is an antibody or anantigen-binding fragment thereof (e.g., any of the antibodies orantigen-binding antibody fragments described herein) are administered toa subject using any of the compositions or devices described herein, theantibody or antigen-binding antibody fragment can penetrate the GItissue of the subject. As used herein, “GI tissue” refers to tissue inthe gastrointestinal (GI) tract, such as tissue in one or more ofduodenum, jejunum, ileum, cecum, ascending colon, transverse colon,descending colon, sigmoid colon, and rectum. In one particularembodiment, GI tissue refers to tissue in the proximal portion of one ormore of duodenum, jejunum, ileum, cecum, ascending colon, transversecolon, descending colon, and sigmoid colon. In one particularembodiment, GI tissue refers to tissue in the distal portion of one ormore of duodenum, jejunum, ileum, cecum, ascending colon, transversecolon, descending colon, and sigmoid colon. The GI tissue may be, forexample, GI tissue proximate to one or more sites of disease.Accordingly, in some embodiments the antibody or antigen-bindingantibody fragment can penetrate the dudodenum tissue proximate to one ormore sites of disease. In some embodiments the antibody orantigen-binding antibody fragment can penetrate the jejunum tissueproximate to one or more sites of disease. In some embodiments theantibody or antigen-binding antibody fragment can penetrate the ileumtissue proximate to one or more sites of disease. In some embodimentsthe antibody or antigen-binding antibody fragment can penetrate thececum tissue proximate to one or more sites of disease. In someembodiments the antibody or antigen-binding antibody fragment canpenetrate the ascending colon tissue proximate to one or more sites ofdisease. In some embodiments the antibody or antigen-binding antibodyfragment can penetrate the transverse colon tissue proximate to one ormore sites of disease. In some embodiments the antibody orantigen-binding antibody fragment can penetrate the descending colontissue proximate to one or more sites of disease. In some embodimentsthe antibody or antigen-binding antibody fragment can penetrate thesigmoid colon tissue proximate to one or more sites of disease. Forexample, an antibody or antigen-binding fragment thereof (e.g., aF(ab′)₂, a Fv, or a scFv) can penetrate one or more (e.g., two, three,or four) of the lumen/superficial mucosa, the lamina propria, thesubmucosa, and the tunica muscularis/serosa. In some embodiments, any ofthe devices or compositions described herein can release a recombinantantibody (e.g., a humanized or fully human antibody, e.g., human orhumanized IgG1, human or humanized IgG2, human or humanized IgG3, humanor humanized IgG4, human or humanized IgA1, human or humanized IgA2,human or humanized IgD, human or humanized IgE, or human or humanizedIgM), which is degraded into an antigen-binding antibody fragment (e.g.,a Fab, a Fv, or a F(ab′)2), which in turn is able to penetrate GI tissue(e.g., one or more (e.g., two, three, or four) of the lumen/superficialmucosa, the lamina propria, the submucosa, and the tunicamuscularis/serosa) of the subject. In some embodiments, the devicereleases an antigen-binding antibody fragment (e.g., any of theantigen-binding antibody fragments described herein).

In some examples, administration of an antibody or an antigen-bindingfragment thereof using any of the compositions or devices describedherein results in penetration (e.g., a detectable level of penetration)of GI tissue (e.g., one or more (e.g., two, three, or four) of thelumen/superficial mucosa, the lamina propria, the submucosa, and thetunica muscularis/serosa) within a time period of about 10 minutes toabout 10 hours, about 10 minutes to about 9 hours, about 10 minutes toabout 8 hours, about 10 minutes to about 7 hours, about 10 minutes toabout 6 hours, about 10 minutes to about 5 hours, about 10 minutes toabout 4.5 hours, about 10 minutes to about 4 hours, about 10 minutes toabout 3.5 hours, about 10 minutes to about 3 hours, about 10 minutes toabout 2.5 hours, about 10 minutes to about 2 hours, about 10 minutes toabout 1.5 hours, about 10 minutes to about 1 hour, about 10 minutes toabout 55 minutes, about 10 minutes to about 50 minutes, about 10 minutesto about 45 minutes, about 10 minutes to about 40 minutes, about 10minutes to about 35 minutes, about 10 minutes to about 30 minutes, about10 minutes to about 25 minutes, about 10 minutes to about 20 minutes,about 10 minutes to about 15 minutes, about 15 minutes to about 10hours, about 15 minutes to about 9 hours, about 15 minutes to about 8hours, about 15 minutes to about 7 hours, about 15 minutes to about 6hours, about 15 minutes to about 5 hours, about 15 minutes to about 4.5hours, about 15 minutes to about 4 hours, about 15 minutes to about 3.5hours, about 15 minutes to about 3 hours, about 15 minutes to about 2.5hours, about 15 minutes to about 2 hours, about 15 minutes to about 1.5hours, about 15 minutes to about 1 hour, about 15 minutes to about 55minutes, about 15 minutes to about 50 minutes, about 15 minutes to about45 minutes, about 15 minutes to about 40 minutes, about 15 minutes toabout 35 minutes, about 15 minutes to about 30 minutes, about 15 minutesto about 25 minutes, about 15 minutes to about 20 minutes, about 20minutes to about 10 hours, about 20 minutes to about 9 hours, about 20minutes to about 8 hours, about 20 minutes to about 7 hours, about 20minutes to about 6 hours, about 20 minutes to about 5 hours, about 20minutes to about 4.5 hours, about 20 minutes to about 4 hours, about 20minutes to about 3.5 hours, about 20 minutes to about 3 hours, about 20minutes to about 2.5 hours, about 20 minutes to about 2 hours, about 20minutes to about 1.5 hours, about 20 minutes to about 1 hour, about 20minutes to about 55 minutes, about 20 minutes to about 50 minutes, about20 minutes to about 45 minutes, about 20 minutes to about 40 minutes,about 20 minutes to about 35 minutes, about 20 minutes to about 30minutes, about 20 minutes to about 25 minutes, about 25 minutes to about10 hours, about 25 minutes to about 9 hours, about 25 minutes to about 8hours, about 25 minutes to about 7 hours, about 25 minutes to about 6hours, about 25 minutes to about 5 hours, about 25 minutes to about 4.5hours, about 25 minutes to about 4 hours, about 25 minutes to about 3.5hours, about 25 minutes to about 3 hours, about 25 minutes to about 2.5hours, about 25 minutes to about 2 hours, about 25 minutes to about 1.5hours, about 25 minutes to about 1 hour, about 25 minutes to about 55minutes, about 25 minutes to about 50 minutes, about 25 minutes to about45 minutes, about 25 minutes to about 40 minutes, about 25 minutes toabout 35 minutes, about 25 minutes to about 30 minutes, about 30 minutesto about 10 hours, about 30 minutes to about 9 hours, about 30 minutesto about 8 hours, about 30 minutes to about 7 hours, about 30 minutes toabout 6 hours, about 30 minutes to about 5 hours, about 30 minutes toabout 4.5 hours, about 30 minutes to about 4 hours, about 30 minutes toabout 3.5 hours, about 30 minutes to about 3 hours, about 30 minutes toabout 2.5 hours, about 30 minutes to about 2 hours, about 30 minutes toabout 1.5 hours, about 30 minutes to about 1 hour, about 30 minutes toabout 55 minutes, about 30 minutes to about 50 minutes, about 30 minutesto about 45 minutes, about 30 minutes to about 40 minutes, about 30minutes to about 35 minutes, about 35 minutes to about 10 hours, about35 minutes to about 9 hours, about 35 minutes to about 8 hours, about 35minutes to about 7 hours, about 35 minutes to about 6 hours, about 35minutes to about 5 hours, about 35 minutes to about 4.5 hours, about 35minutes to about 4 hours, about 35 minutes to about 3.5 hours, about 35minutes to about 3 hours, about 35 minutes to about 2.5 hours, about 35minutes to about 2 hours, about 35 minutes to about 1.5 hours, about 35minutes to about 1 hour, about 35 minutes to about 55 minutes, about 35minutes to about 50 minutes, about 35 minutes to about 45 minutes, about35 minutes to about 40 minutes, about 40 minutes to about 10 hours,about 40 minutes to about 9 hours, about 40 minutes to about 8 hours,about 40 minutes to about 7 hours, about 40 minutes to about 6 hours,about 40 minutes to about 5 hours, about 40 minutes to about 4.5 hours,about 40 minutes to about 4 hours, about 40 minutes to about 3.5 hours,about 40 minutes to about 3 hours, about 40 minutes to about 2.5 hours,about 40 minutes to about 2 hours, about 40 minutes to about 1.5 hours,about 40 minutes to about 1 hour, about 40 minutes to about 55 minutes,about 40 minutes to about 50 minutes, about 40 minutes to about 45minutes, about 45 minutes to about 10 hours, about 45 minutes to about 9hours, about 45 minutes to about 8 hours, about 45 minutes to about 7hours, about 45 minutes to about 6 hours, about 45 minutes to about 5hours, about 45 minutes to about 4.5 hours, about 45 minutes to about 4hours, about 45 minutes to about 3.5 hours, about 45 minutes to about 3hours, about 45 minutes to about 2.5 hours, about 45 minutes to about 2hours, about 45 minutes to about 1.5 hours, about 45 minutes to about 1hour, about 45 minutes to about 55 minutes, about 45 minutes to about 50minutes, about 50 minutes to about 10 hours, about 50 minutes to about 9hours, about 50 minutes to about 8 hours, about 50 minutes to about 7hours, about 50 minutes to about 6 hours, about 50 minutes to about 5hours, about 50 minutes to about 4.5 hours, about 50 minutes to about 4hours, about 50 minutes to about 3.5 hours, about 50 minutes to about 3hours, about 50 minutes to about 2.5 hours, about 50 minutes to about 2hours, about 50 minutes to about 1.5 hours, about 50 minutes to about 1hour, about 50 minutes to about 55 minutes, about 55 minutes to about 10hours, about 55 minutes to about 9 hours, about 55 minutes to about 8hours, about 55 minutes to about 7 hours, about 55 minutes to about 6hours, about 55 minutes to about 5 hours, about 55 minutes to about 4.5hours, about 55 minutes to about 4 hours, about 55 minutes to about 3.5hours, about 55 minutes to about 3 hours, about 55 minutes to about 2.5hours, about 55 minutes to about 2 hours, about 55 minutes to about 1.5hours, about 55 minutes to about 1 hour, about 1 hour to about 10 hours,about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour toabout 5 hours, about 1 hour to about 4.5 hours, about 1 hour to about 4hours, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours,about 1 hour to about 2.5 hours, about 1 hour to about 2 hours, about 1hour to about 1.5 hours, about 1.5 hours to about 10 hours, about 1.5hours to about 9 hours, about 1.5 hours to about 8 hours, about 1.5hours to about 7 hours, about 1.5 hours to about 6 hours, about 1.5hours to about 5 hours, about 1.5 hours to about 4.5 hours, about 1.5hours to about 4 hours, about 1.5 hours to about 3.5 hours, about 1.5hours to about 3 hours, about 1.5 hours to about 2.5 hours, about 1.5hours to about 2 hours, about 2 hours to about 10 hours, about 2 hoursto about 9 hours, about 2 hours to about 8 hours, about 2 hours to about7 hours, about 2 hours to about 6 hours, about 2 hours to about 5 hours,about 2 hours to about 4.5 hours, about 2 hours to about 4 hours, about2 hours to about 3.5 hours, about 2 hours to about 3 hours, about 2hours to about 2.5 hours, about 2.5 hours to about 10 hours, about 2.5hours to about 9 hours, about 2.5 hours to about 8 hours, about 2.5hours to about 7 hours, about 2.5 hours to about 6 hours, about 2.5hours to about 5 hours, about 2.5 hours to about 4.5 hours, about 2.5hours to about 4 hours, about 2.5 hours to about 3.5 hours, about 2.5hours to about 3 hours, about 3 hours to about 10 hours, about 3 hoursto about 9 hours, about 3 hours to about 8 hours, about 3 hours to about7 hours, about 3 hours to about 6 hours, about 3 hours to about 5 hours,about 3 hours to about 4.5 hours, about 3 hours to about 4 hours, about3 hours to about 3.5 hours, about 3.5 hours to about 10 hours, about 3.5hours to about 9 hours, about 3.5 hours to about 8 hours, about 3.5hours to about 7 hours, about 3.5 hours to about 6 hours, about 3.5hours to about 5 hours, about 3.5 hours to about 4.5 hours, about 3.5hours to about 4 hours, about 4 hours to about 10 hours, about 4 hoursto about 9 hours, about 4 hours to about 8 hours, about 4 hours to about7 hours, about 4 hours to about 6 hours, about 4 hours to about 5 hours,about 4 hours to about 4.5 hours, about 4.5 hours to about 10 hours,about 4.5 hours to about 9 hours, about 4.5 hours to about 8 hours,about 4.5 hours to about 7 hours, about 4.5 hours to about 6 hours,about 4.5 hours to about 5 hours, about 5 hours to about 10 hours, about5 hours to about 9 hours, about 5 hours to about 8 hours, about 5 hoursto about 7 hours, about 5 hours to about 6 hours, about 6 hours to about10 hours, about 6 hours to about 9 hours, about 6 hours to about 8hours, about 6 hours to about 7 hours, about 7 hours to about 10 hours,about 7 hours to about 9 hours, about 7 hours to about 8 hours, about 8hours to about 10 hours, about 8 hours to about 9 hours, or about 9hours to about 10 hours. Penetration of GI tissue by an antibody or anantigen-binding antibody fragment can be detected by administering alabeled antibody or labeled antigen-binding antibody fragment, andperforming imaging on the subject (e.g., ultrasound, computedtomography, or magnetic resonance imaging). For example, the label canbe a radioisotope, a heavy metal, a fluorophore, or a luminescent agent(e.g., any suitable radioisotopes, heavy metals, fluorophores, orluminescent agents used for imaging known in the art).

While not wishing to be bound to a particular theory, the inventorscontemplate that at or near the site of release a concentration gradientof the TLR agonist is generated in the mucosa, and that administrationof an TLR agonist using a device as described herein advantageouslyresults in a “reverse” concentration gradient when compared to theconcentration gradient resulting from systemic administration. In such“reverse” concentration gradient, the drug concentration is highest fromsuperficial to deep with respect to the mucosal surface. Systemicadministration instead typically results in concentrations of the drugbeing highest from deep to superficial. A “reverse” concentrationgradient as described above aligns more favorably with thepathophysiology of IBD.

In some embodiments, administration of an antibody or an antigen-bindingantibody fragment can provide for treatment (e.g., a reduction in thenumber, severity, and/or duration of one or more symptoms of any of thedisorders described herein in a subject) for a time period of betweenabout 1 hour to about 30 days, about 1 hour to about 28 days, about 1hour to about 26 days, about 1 hour to about 24 days, about 1 hour toabout 22 days, about 1 hour to about 20 days, about 1 hour to about 18days, about 1 hour to about 16 days, about 1 hour to about 14 days,about 1 hour to about 12 days, about 1 hour to about 10 days, about 1hour to about 8 days, about 1 hour to about 6 days, about 1 hour toabout 5 days, about 1 hour to about 4 days, about 1 hour to about 3days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1hour to about 12 hours, about 1 hour to about 6 hours, about 1 hour toabout 3 hours, about 3 hours to about 30 days, about 3 hours to about 28days, about 3 hours to about 26 days, about 3 hours to about 24 days,about 3 hours to about 22 days, about 3 hours to about 20 days, about 3hours to about 18 days, about 3 hours to about 16 days, about 3 hours toabout 14 days, about 3 hours to about 12 days, about 3 hours to about 10days, about 3 hours to about 8 days, about 3 hours to about 6 days,about 3 hours to about 5 days, about 3 hours to about 4 days, about 3hours to about 3 days, about 3 hours to about 2 days, about 3 hours toabout 1 day, about 3 hours to about 12 hours, about 3 hours to about 6hours, about 6 hours to about 30 days, about 6 hours to about 28 days,about 6 hours to about 26 days, about 6 hours to about 24 days, about 6hours to about 22 days, about 6 hours to about 20 days, about 6 hours toabout 18 days, about 6 hours to about 16 days, about 6 hours to about 14days, about 6 hours to about 12 days, about 6 hours to about 10 days,about 6 hours to about 8 days, about 6 hours to about 6 days, about 6hours to about 5 days, about 6 hours to about 4 days, about 6 hours toabout 3 days, about 6 hours to about 2 days, about 6 hours to about 1day, about 6 hours to about 12 hours, about 12 hours to about 30 days,about 12 hours to about 28 days, about 12 hours to about 26 days, about12 hours to about 24 days, about 12 hours to about 22 days, about 12hours to about 20 days, about 12 hours to about 18 days, about 12 hoursto about 16 days, about 12 hours to about 14 days, about 12 hours toabout 12 days, about 12 hours to about 10 days, about 12 hours to about8 days, about 12 hours to about 6 days, about 12 hours to about 5 days,about 12 hours to about 4 days, about 12 hours to about 3 days, about 12hours to about 2 days, about 12 hours to about 1 day, about 1 day toabout 30 days, about 1 day to about 28 days, about 1 day to about 26days, about 1 day to about 24 days, about 1 day to about 22 days, about1 day to about 20 days, about 1 day to about 18 days, about 1 day toabout 16 days, about 1 day to about 14 days, about 1 day to about 12days, about 1 day to about 10 days, about 1 day to about 8 days, about 1day to about 6 days, about 1 day to about 5 days, about 1 day to about 4days, about 1 day to about 3 days, about 1 day to about 2 days, about 2days to about 30 days, about 2 days to about 28 days, about 2 days toabout 26 days, about 2 days to about 24 days, about 2 days to about 22days, about 2 days to about 20 days, about 2 days to about 18 days,about 2 days to about 16 days, about 2 days to about 14 days, about 2days to about 12 days, about 2 days to about 10 days, about 2 days toabout 8 days, about 2 days to about 6 days, about 2 days to about 5days, about 2 days to about 4 days, about 2 days to about 3 days, about3 days to about 30 days, about 3 days to about 28 days, about 3 days toabout 26 days, about 3 days to about 24 days, about 3 days to about 22days, about 3 days to about 20 days, about 3 days to about 18 days,about 3 days to about 16 days, about 3 days to about 14 days, about 3days to about 12 days, about 3 days to about 10 days, about 3 days toabout 8 days, about 3 days to about 6 days, about 3 days to about 5days, about 3 days to about 4 days, about 4 days to about 30 days, about4 days to about 28 days, about 4 days to about 26 days, about 4 days toabout 24 days, about 4 days to about 22 days, about 4 days to about 20days, about 4 days to about 18 days, about 4 days to about 16 days,about 4 days to about 14 days, about 4 days to about 12 days, about 4days to about 10 days, about 4 days to about 8 days, about 4 days toabout 6 days, about 4 days to about 5 days, about 5 days to about 30days, about 5 days to about 28 days, about 5 days to about 26 days,about 5 days to about 24 days, about 5 days to about 22 days, about 5days to about 20 days, about 5 days to about 18 days, about 5 days toabout 16 days, about 5 days to about 14 days, about 5 days to about 12days, about 5 days to about 10 days, about 5 days to about 8 days, about5 days to about 6 days, about 6 days to about 30 days, about 6 days toabout 28 days, about 6 days to about 26 days, about 6 days to about 24days, about 6 days to about 22 days, about 6 days to about 20 days,about 6 days to about 18 days, about 6 days to about 16 days, about 6days to about 14 days, about 6 days to about 12 days, about 6 days toabout 10 days, about 6 days to about 8 days, about 8 days to about 30days, about 8 days to about 28 days, about 8 days to about 26 days,about 8 days to about 24 days, about 8 days to about 22 days, about 8days to about 20 days, about 8 days to about 18 days, about 8 days toabout 16 days, about 8 days to about 14 days, about 8 days to about 12days, about 8 days to about 10 days, about 10 days to about 30 days,about 10 days to about 28 days, about 10 days to about 26 days, about 10days to about 24 days, about 10 days to about 22 days, about 10 days toabout 20 days, about 10 days to about 18 days, about 10 days to about 16days, about 10 days to about 14 days, about 10 days to about 12 days,about 12 days to about 30 days, about 12 days to about 28 days, about 12days to about 26 days, about 12 days to about 24 days, about 12 days toabout 22 days, about 12 days to about 20 days, about 12 days to about 18days, about 12 days to about 16 days, about 12 days to about 14 days,about 14 days to about 30 days, about 14 days to about 28 days, about 14days to about 26 days, about 14 days to about 24 days, about 14 days toabout 22 days, about 14 days to about 20 days, about 14 days to about 18days, about 14 days to about 16 days, about 16 days to about 30 days,about 16 days to about 28 days, about 16 days to about 26 days, about 16days to about 24 days, about 16 days to about 22 days, about 16 days toabout 20 days, about 16 days to about 18 days, about 18 days to about 30days, about 18 days to about 28 days, about 18 days to about 26 days,about 18 days to about 24 days, about 18 days to about 22 days, about 18days to about 20 days, about 20 days to about 30 days, about 20 days toabout 28 days, about 20 days to about 26 days, about 20 days to about 24days, about 20 days to about 22 days, about 22 days to about 30 days,about 22 days to about 28 days, about 22 days to about 26 days, about 22days to about 24 days, about 24 days to about 30 days, about 24 days toabout 28 days, about 24 days to about 26 days, about 26 days to about 30days, about 26 days to about 28 days, or about 28 days to about 30 daysin a subject following first administration of an antibody orantigen-binding antibody fragment using any of the compositions ordevices described herein. Non-limiting examples of symptoms of a diseasedescribed herein are described below.

For example, treatment can result in a decrease (e.g., about 1% to about99% decrease, about 1% to about 95% decrease, about 1% to about 90%decrease, about 1% to about 85% decrease, about 1% to about 80%decrease, about 1% to about 75% decrease, about 1% to about 70%decrease, about 1% to about 65% decrease, about 1% to about 60%decrease, about 1% to about 55% decrease, about 1% to about 50%decrease, about 1% to about 45% decrease, about 1% to about 40%decrease, about 1% to about 35% decrease, about 1% to about 30%decrease, about 1% to about 25% decrease, about 1% to about 20%decrease, about 1% to about 15% decrease, about 1% to about 10%decrease, about 1% to about 5% decrease, about 5% to about 99% decrease,about 5% to about 95% decrease, about 5% to about 90% decrease, about 5%to about 85% decrease, about 5% to about 80% decrease, about 5% to about75% decrease, about 5% to about 70% decrease, about 5% to about 65%decrease, about 5% to about 60% decrease, about 5% to about 55%decrease, about 5% to about 50% decrease, about 5% to about 45%decrease, about 5% to about 40% decrease, about 5% to about 35%decrease, about 5% to about 30% decrease, about 5% to about 25%decrease, about 5% to about 20% decrease, about 5% to about 15%decrease, about 5% to about 10% decrease, about 10% to about 99%decrease, about 10% to about 95% decrease, about 10% to about 90%decrease, about 10% to about 85% decrease, about 10% to about 80%decrease, about 10% to about 75% decrease, about 10% to about 70%decrease, about 10% to about 65% decrease, about 10% to about 60%decrease, about 10% to about 55% decrease, about 10% to about 50%decrease, about 10% to about 45% decrease, about 10% to about 40%decrease, about 10% to about 35% decrease, about 10% to about 30%decrease, about 10% to about 25% decrease, about 10% to about 20%decrease, about 10% to about 15% decrease, about 15% to about 99%decrease, about 15% to about 95% decrease, about 15% to about 90%decrease, about 15% to about 85% decrease, about 15% to about 80%decrease, about 15% to about 75% decrease, about 15% to about 70%decrease, about 15% to about 65% decrease, about 15% to about 60%decrease, about 15% to about 55% decrease, about 15% to about 50%decrease, about 15% to about 45% decrease, about 15% to about 40%decrease, about 15% to about 35% decrease, about 15% to about 30%decrease, about 15% to about 25% decrease, about 15% to about 20%decrease, about 20% to about 99% decrease, about 20% to about 95%decrease, about 20% to about 90% decrease, about 20% to about 85%decrease, about 20% to about 80% decrease, about 20% to about 75%decrease, about 20% to about 70% decrease, about 20% to about 65%decrease, about 20% to about 60% decrease, about 20% to about 55%decrease, about 20% to about 50% decrease, about 20% to about 45%decrease, about 20% to about 40% decrease, about 20% to about 35%decrease, about 20% to about 30% decrease, about 20% to about 25%decrease, about 25% to about 99% decrease, about 25% to about 95%decrease, about 25% to about 90% decrease, about 25% to about 85%decrease, about 25% to about 80% decrease, about 25% to about 75%decrease, about 25% to about 70% decrease, about 25% to about 65%decrease, about 25% to about 60% decrease, about 25% to about 55%decrease, about 25% to about 50% decrease, about 25% to about 45%decrease, about 25% to about 40% decrease, about 25% to about 35%decrease, about 25% to about 30% decrease, about 30% to about 99%decrease, about 30% to about 95% decrease, about 30% to about 90%decrease, about 30% to about 85% decrease, about 30% to about 80%decrease, about 30% to about 75% decrease, about 30% to about 70%decrease, about 30% to about 65% decrease, about 30% to about 60%decrease, about 30% to about 55% decrease, about 30% to about 50%decrease, about 30% to about 45% decrease, about 30% to about 40%decrease, about 30% to about 35% decrease, about 35% to about 99%decrease, about 35% to about 95% decrease, about 35% to about 90%decrease, about 35% to about 85% decrease, about 35% to about 80%decrease, about 35% to about 75% decrease, about 35% to about 70%decrease, about 35% to about 65% decrease, about 35% to about 60%decrease, about 35% to about 55% decrease, about 35% to about 50%decrease, about 35% to about 45% decrease, about 35% to about 40%decrease, about 40% to about 99% decrease, about 40% to about 95%decrease, about 40% to about 90% decrease, about 40% to about 85%decrease, about 40% to about 80% decrease, about 40% to about 75%decrease, about 40% to about 70% decrease, about 40% to about 65%decrease, about 40% to about 60% decrease, about 40% to about 55%decrease, about 40% to about 50% decrease, about 40% to about 45%decrease, about 45% to about 99% decrease, about 45% to about 95%decrease, about 45% to about 90% decrease, about 45% to about 85%decrease, about 45% to about 80% decrease, about 45% to about 75%decrease, about 45% to about 70% decrease, about 45% to about 65%decrease, about 45% to about 60% decrease, about 45% to about 55%decrease, about 45% to about 50% decrease, about 50% to about 99%decrease, about 50% to about 95% decrease, about 50% to about 90%decrease, about 50% to about 85% decrease, about 50% to about 80%decrease, about 50% to about 75% decrease, about 50% to about 70%decrease, about 50% to about 65% decrease, about 50% to about 60%decrease, about 50% to about 55% decrease, about 55% to about 99%decrease, about 55% to about 95% decrease, about 55% to about 90%decrease, about 55% to about 85% decrease, about 55% to about 80%decrease, about 55% to about 75% decrease, about 55% to about 70%decrease, about 55% to about 65% decrease, about 55% to about 60%decrease, about 60% to about 99% decrease, about 60% to about 95%decrease, about 60% to about 90% decrease, about 60% to about 85%decrease, about 60% to about 80% decrease, about 60% to about 75%decrease, about 60% to about 70% decrease, about 60% to about 65%decrease, about 65% to about 99% decrease, about 65% to about 95%decrease, about 65% to about 90% decrease, about 65% to about 85%decrease, about 65% to about 80% decrease, about 65% to about 75%decrease, about 65% to about 70% decrease, about 70% to about 99%decrease, about 70% to about 95% decrease, about 70% to about 90%decrease, about 70% to about 85% decrease, about 70% to about 80%decrease, about 70% to about 75% decrease, about 75% to about 99%decrease, about 75% to about 95% decrease, about 75% to about 90%decrease, about 75% to about 85% decrease, about 75% to about 80%decrease, about 80% to about 99% decrease, about 80% to about 95%decrease, about 80% to about 90% decrease, about 80% to about 85%decrease, about 85% to about 99% decrease, about 85% to about 95%decrease, about 85% to about 90% decrease, about 90% to about 99%decrease, about 90% to about 95% decrease, or about 95% to about 99%decrease) in one or more (e.g., two, three, four, five, six, seven,eight, or nine) of: the level of interferon-γ in GI tissue, the level ofIL-1β in GI tissue, the level of IL-6 in GI tissue, the level of IL-22in GI tissue, the level of IL-17A in the GI tissue, the level of TNFα inGI tissue, the level of IL-2 in GI tissue, and endoscopy score in asubject (e.g., as compared to the level in the subject prior totreatment or compared to a subject or population of subjects having asimilar disease but receiving a placebo or a different treatment) (e.g.,for a time period of between about 1 hour to about 30 days (e.g., or anyof the subranges herein) following the first administration of anantibody or antigen-binding antibody fragment using any of thecompositions or devices described herein. Exemplary methods fordetermining the endoscopy score are described herein and other methodsfor determining the endoscopy score are known in the art. Exemplarymethods for determining the levels of interferon-γ, IL-1β, IL-6, IL-22,IL-17A, TNFα, and IL-2 are described herein. Additional methods fordetermining the levels of these cytokines are known in the art.

In some examples, treatment can result in an increase (e.g., about 1% toabout 500% increase, about 1% to about 400% increase, about 1% to about300% increase, about 1% to about 200% increase, about 1% to about 150%increase, about 1% to about 100% increase, about 1% to about 90%increase, about 1% to about 80% increase, about 1% to about 70%increase, about 1% to about 60% increase, about 1% to about 50%increase, about 1% to about 40% increase, about 1% to about 30%increase, about 1% to about 20% increase, about 1% to about 10%increase, a 10% to about 500% increase, about 10% to about 400%increase, about 10% to about 300% increase, about 10% to about 200%increase, about 10% to about 150% increase, about 10% to about 100%increase, about 10% to about 90% increase, about 10% to about 80%increase, about 10% to about 70% increase, about 10% to about 60%increase, about 10% to about 50% increase, about 10% to about 40%increase, about 10% to about 30% increase, about 10% to about 20%increase, about 20% to about 500% increase, about 20% to about 400%increase, about 20% to about 300% increase, about 20% to about 200%increase, about 20% to about 150% increase, about 20% to about 100%increase, about 20% to about 90% increase, about 20% to about 80%increase, about 20% to about 70% increase, about 20% to about 60%increase, about 20% to about 50% increase, about 20% to about 40%increase, about 20% to about 30% increase, about 30% to about 500%increase, about 30% to about 400% increase, about 30% to about 300%increase, about 30% to about 200% increase, about 30% to about 150%increase, about 30% to about 100% increase, about 30% to about 90%increase, about 30% to about 80% increase, about 30% to about 70%increase, about 30% to about 60% increase, about 30% to about 50%increase, about 30% to about 40% increase, about 40% to about 500%increase, about 40% to about 400% increase, about 40% to about 300%increase, about 40% to about 200% increase, about 40% to about 150%increase, about 40% to about 100% increase, about 40% to about 90%increase, about 40% to about 80% increase, about 40% to about 70%increase, about 40% to about 60% increase, about 40% to about 50%increase, about 50% to about 500% increase, about 50% to about 400%increase, about 50% to about 300% increase, about 50% to about 200%increase, about 50% to about 150% increase, about 50% to about 100%increase, about 50% to about 90% increase, about 50% to about 80%increase, about 50% to about 70% increase, about 50% to about 60%increase, about 60% to about 500% increase, about 60% to about 400%increase, about 60% to about 300% increase, about 60% to about 200%increase, about 60% to about 150% increase, about 60% to about 100%increase, about 60% to about 90% increase, about 60% to about 80%increase, about 60% to about 70% increase, about 70% to about 500%increase, about 70% to about 400% increase, about 70% to about 300%increase, about 70% to about 200% increase, about 70% to about 150%increase, about 70% to about 100% increase, about 70% to about 90%increase, about 70% to about 80% increase, about 80% to about 500%increase, about 80% to about 400% increase, about 80% to about 300%increase, about 80% to about 200% increase, about 80% to about 150%increase, about 80% to about 100% increase, about 80% to about 90%increase, about 90% to about 500% increase, about 90% to about 400%increase, about 90% to about 300% increase, about 90% to about 200%increase, about 90% to about 150% increase, about 90% to about 100%increase, about 100% to about 500% increase, about 100% to about 400%increase, about 100% to about 300% increase, about 100% to about 200%increase, about 100% to about 150% increase, about 150% to about 500%increase, about 150% to about 400% increase, about 150% to about 300%increase, about 150% to about 200% increase, about 200% to about 500%increase, about 200% to about 400% increase, about 200% to about 300%increase, about 300% to about 500% increase, about 300% to about 400%increase, or about 400% to about 500% increase) in one or both of stoolconsistency score and weight of a subject (e.g., as compared to thelevel in the subject prior to treatment or compared to a subject orpopulation of subjects having a similar disease but receiving a placeboor a different treatment) (e.g., for a time period of between about 1hour to about 30 days (e.g., or any of the subranges herein) followingthe first administration of an antibody or antigen-binding antibodyfragment using any of the compositions or devices described herein.Exemplary methods for determining stool consistency score are describedherein. Additional methods for determining a stool consistency score areknown in the art.

In some examples, administration of an antibody or an antigen-bindingantibody fragment using any of the devices or compositions describedherein can result in a ratio of GI tissue concentration of the antibodyor the antigen-binding antibody fragment to the blood, serum, or plasmaconcentration of the antibody or the antigen-binding antibody fragmentof, e.g., about 2.8 to about 6.0, about 2.8 to about 5.8, about 2.8 toabout 5.6, about 2.8 to about 5.4, about 2.8 to about 5.2, about 2.8 toabout 5.0, about 2.8 to about 4.8, about 2.8 to about 4.6, about 2.8 toabout 4.4, about 2.8 to about 4.2, about 2.8 to about 4.0, about 2.8 toabout 3.8, about 2.8 to about 3.6, about 2.8 to about 3.4, about 2.8 toabout 3.2, about 2.8 to about 3.0, about 3.0 to about 6.0, about 3.0 toabout 5.8, about 3.0 to about 5.6, about 3.0 to about 5.4, about 3.0 toabout 5.2, about 3.0 to about 5.0, about 3.0 to about 4.8, about 3.0 toabout 4.6, about 3.0 to about 4.4, about 3.0 to about 4.2, about 3.0 toabout 4.0, about 3.0 to about 3.8, about 3.0 to about 3.6, about 3.0 toabout 3.4, about 3.0 to about 3.2, about 3.2 to about 6.0, about 3.2 toabout 5.8, about 3.2 to about 5.6, about 3.2 to about 5.4, about 3.2 toabout 5.2, about 3.2 to about 5.0, about 3.2 to about 4.8, about 3.2 toabout 4.6, about 3.2 to about 4.4, about 3.2 to about 4.2, about 3.2 toabout 4.0, about 3.2 to about 3.8, about 3.2 to about 3.6, about 3.2 toabout 3.4, about 3.4 to about 6.0, about 3.4 to about 5.8, about 3.4 toabout 5.6, about 3.4 to about 5.4, about 3.4 to about 5.2, about 3.4 toabout 5.0, about 3.4 to about 4.8, about 3.4 to about 4.6, about 3.4 toabout 4.4, about 3.4 to about 4.2, about 3.4 to about 4.0, about 3.4 toabout 3.8, about 3.4 to about 3.6, about 3.6 to about 6.0, about 3.6 toabout 5.8, about 3.6 to about 5.6, about 3.6 to about 5.4, about 3.6 toabout 5.2, about 3.6 to about 5.0, about 3.6 to about 4.8, about 3.6 toabout 4.6, about 3.6 to about 4.4, about 3.6 to about 4.2, about 3.6 toabout 4.0, about 3.6 to about 3.8, about 3.8 to about 6.0, about 3.8 toabout 5.8, about 3.8 to about 5.6, about 3.8 to about 5.4, about 3.8 toabout 5.2, about 3.8 to about 5.0, about 3.8 to about 4.8, about 3.8 toabout 4.6, about 3.8 to about 4.4, about 3.8 to about 4.2, about 3.8 toabout 4.0, about 4.0 to about 6.0, about 4.0 to about 5.8, about 4.0 toabout 5.6, about 4.0 to about 5.4, about 4.0 to about 5.2, about 4.0 toabout 5.0, about 4.0 to about 4.8, about 4.0 to about 4.6, about 4.0 toabout 4.4, about 4.0 to about 4.2, about 4.2 to about 6.0, about 4.2 toabout 5.8, about 4.2 to about 5.6, about 4.2 to about 5.4, about 4.2 toabout 5.2, about 4.2 to about 5.0, about 4.2 to about 4.8, about 4.2 toabout 4.6, about 4.2 to about 4.4, about 4.4 to about 6.0, about 4.4 toabout 5.8, about 4.4 to about 5.6, about 4.4 to about 5.4, about 4.4 toabout 5.2, about 4.4 to about 5.0, about 4.4 to about 4.8, about 4.4 toabout 4.6, about 4.6 to about 6.0, about 4.6 to about 5.8, about 4.6 toabout 5.6, about 4.6 to about 5.4, about 4.6 to about 5.2, about 4.6 toabout 5.0, about 4.6 to about 4.8, about 4.8 to about 6.0, about 4.8 toabout 5.8, about 4.8 to about 5.6, about 4.8 to about 5.4, about 4.8 toabout 5.2, about 4.8 to about 5.0, about 5.0 to about 6.0, about 5.0 toabout 5.8, about 5.0 to about 5.6, about 5.0 to about 5.4, about 5.0 toabout 5.2, about 5.2 to about 6.0, about 5.2 to about 5.8, about 5.2 toabout 5.6, about 5.2 to about 5.4, about 5.4 to about 6.0, about 5.4 toabout 5.8, about 5.4 to about 5.6, about 5.6 to about 6.0, about 5.6 toabout 5.8, or about 5.8 to about 6.0. Accordingly, in some embodiments,a method of treatment disclosed herein can include determining the ratioof the level of the TLR agonist in the GI tissue to the level of the TLRagonist in the blood, serum, or plasma of a subject at substantially thesame time point following administration of the device is about 2.8 toabout 6.0. Exemplary methods for measuring the concentration of anantibody or an antigen-binding antibody fragment in the plasma or the GItissue of a subject are described herein. Additional methods formeasuring the concentration of an antibody or an antigen-bindingantibody fragment in the plasma or the GI tissue of a subject are knownin the art.

Accordingly, in some embodiments, a method of treatment disclosed hereinincludes determining the level of the TLR agonist in the GI tissue(e.g., one or more of any of the exemplary GI tissues described herein).In some embodiments, a method of treatment disclosed herein can includedetermining the level of TLR agonist in one or more (e.g., two, three,or four) of the lumen/superficial mucosa, the lamina propria, thesubmucosa, and the tunica muscularis/serosa.

In some embodiments, a method of treatment disclosed herein includesdetermining that the level of the TLR agonist in the GI tissue (e.g.,one or more of any of the exemplary types of GI tissues describedherein) at a time point following administration of the device is higherthan the level of the TLR agonist in the GI tissue at substantially thesame time point following systemic administration of an equal amount ofthe TLR agonist. In some embodiments, a method of treatment disclosedherein can include determining that the level of the TLR agonist in oneor more (e.g., two, three, or four) of the lumen/superficial mucosa, thelamina propria, the submucosa, and the tunica muscularis/serosa at atime point following administration of the device is higher than thelevel of the TLR agonist in one or more (e.g., two, three, or four) ofthe lumen/superficial mucosa, the lamina propria, the submucosa, and thetunica muscularis/serosa at substantially the same time point followingsystemic administration of an equal amount of the TLR agonist.

In some embodiments, a method of treatment disclosed herein includesdetermining the level of TLR agonist in the feces of the subject. Insome embodiments, a method of treatment disclosed herein includesdetermining the level of TLR agonist in the GI tissue, e.g., in one ormore (e.g., two, three, or four) of the lumen/superficial mucosa, thelamina propria, the submucosa, and the tunica muscularis/serosa within atime period of about 10 minutes to about 10 hours followingadministration of the device.

In some embodiments, a method of treatment as disclosed herein comprisesdetermining the level of the TLR agonist at the location of diseasefollowing administration of the device.

In some embodiments, a method of treatment as disclosed herein comprisesdetermining that the level of TLR agonist at the location of disease ata time point following administration of the device is higher than thelevel of the TLR agonist at the same location of disease atsubstantially the same time point following systemic administration ofan equal amount of the TLR agonist.

In some embodiments, a method of treatment as disclosed herein comprisesdetermining that the level of TLR agonist in plasma in a subject at atime point following administration of the device is lower than thelevel of the TLR agonist in plasma in a subject at substantially thesame time point following systemic administration of an equal amount ofthe TLR agonist.

In some embodiments, a method of treatment as disclosed herein comprisesdetermining the level of the TLR agonist in the tissue of the subjectwithin a time period of about 10 minutes to 10 hours followingadministration of the device.

Some examples of any of the methods described herein can, e.g., resultin a selective suppression of a local inflammatory response (e.g., aninflammatory response in local GI tissue), while maintaining thesystemic immune response (e.g., blood). The GI tissue may be, forexample, GI tissue proximate to one or more sites of disease. FAs usedherein, “GI content” refers to the content of the gastrointestinal (GI)tract, such as the content of one or more of duodenum, jejunum, ileum,cecum, ascending colon, transverse colon, descending colon, sigmoidcolon, and rectum, more particularly of the proximal portion of one ormore of duodenum, jejunum, ileum, cecum, ascending colon, transversecolon, descending colon, and sigmoid colon, or of the distal portion ofone or more of duodenum, jejunum, ileum, cecum, ascending colon,transverse colon, descending colon, and sigmoid colon. Accordingly, insome embodiments, the methods described herein can result in a selectivesuppression of the inflammatory response in the dudodenum tissueproximate to one or more sites of disease, while maintaining thesystemic immune response. In some embodiments, the methods describedherein can result in a selective suppression of the inflammatoryresponse in the jejunum tissue proximate to one or more sites ofdisease, while maintaining the systemic immune response. In someembodiments, the methods described herein can result in a selectivesuppression of the inflammatory response in the ileum tissue proximateto one or more sites of disease, while maintaining the systemic immuneresponse. In some embodiments, the methods described herein can resultin a selective suppression of the inflammatory response in the cecumtissue proximate to one or more sites of disease, while maintaining thesystemic immune response. In some embodiments, the methods describedherein can result in a selective suppression of the inflammatoryresponse in the ascending colon tissue proximate to one or more sites ofdisease, while maintaining the systemic immune response. In someembodiments, the methods described herein can result in a selectivesuppression of the inflammatory response in the transverse colon tissueproximate to one or more sites of disease, while maintaining thesystemic immune response. In some embodiments, the methods describedherein can result in a selective suppression of the inflammatoryresponse in the descending colon tissue proximate to one or more sitesof disease, while maintaining the systemic immune response. In someembodiments, the methods described herein can result in a selectivesuppression of the inflammatory response in the sigmoid colon tissueproximate to one or more sites of disease, while maintaining thesystemic immune response. In some examples, the methods described hereincan result in a 1% increase to 500% increase (e.g., a 1% increase to450% increase, a 1% increase to 400% increase, a 1% increase to 350%increase, a 1% increase to 300% increase, a 1% increase to 250%increase, a 1% increase to 200% increase, a 1% increase to 190%increase, a 1% increase to 180% increase, a 1% increase to 170%increase, a 1% increase to 160% increase, a 1% increase to 150%increase, a 1% increase to 140% increase, a 1% increase to 130%increase, a 1% increase to 120% increase, a 1% increase to 110%increase, a 1% increase to 100% increase, a 1% increase to 90% increase,a 1% increase to 80% increase, a 1% increase to 70% increase, a 1%increase to 60% increase, a 1% increase to 50% increase, a 1% increaseto 40% increase, a 1% increase to 30% increase, a 1% increase to 25%increase, a 1% increase to 20% increase, a 1% increase to 15% increase,a 1% increase to 10% increase, a 1% increase to 5% increase, a 5%increase to 500% increase, a 5% increase to 450% increase, a 5% increaseto 400% increase, a 5% increase to 350% increase, a 5% increase to 300%increase, a 5% increase to 250% increase, a 5% increase to 200%increase, a 5% increase to 190% increase, a 5% increase to 180%increase, a 5% increase to 170% increase, a 5% increase to 160%increase, a 5% increase to 150% increase, a 5% increase to 140%increase, a 5% increase to 130% increase, a 5% increase to 120%increase, a 5% increase to 110% increase, a 5% increase to 100%increase, a 5% increase to 90% increase, a 5% increase to 80% increase,a 5% increase to 70% increase, a 5% increase to 60% increase, a 5%increase to 50% increase, a 5% increase to 40% increase, a 5% increaseto 30% increase, a 5% increase to 25% increase, a 5% increase to 20%increase, a 5% increase to 15% increase, a 5% increase to 10% increase,a 10% increase to 500% increase, a 10% increase to 450% increase, a 10%increase to 400% increase, a 10% increase to 350% increase, a 10%increase to 300% increase, a 10% increase to 250% increase, a 10%increase to 200% increase, a 10% increase to 190% increase, a 10%increase to 180% increase, a 10% increase to 170% increase, a 10%increase to 160% increase, a 10% increase to 150% increase, a 10%increase to 140% increase, a 10% increase to 130% increase, a 10%increase to 120% increase, a 10% increase to 110% increase, a 10%increase to 100% increase, a 10% increase to 90% increase, a 10%increase to 80% increase, a 10% increase to 70% increase, a 10% increaseto 60% increase, a 10% increase to 50% increase, a 10% increase to 40%increase, a 10% increase to 30% increase, a 10% increase to 25%increase, a 10% increase to 20% increase, a 10% increase to 15%increase, a 15% increase to 500% increase, a 15% increase to 450%increase, a 15% increase to 400% increase, a 15% increase to 350%increase, a 15% increase to 300% increase, a 15% increase to 250%increase, a 15% increase to 200% increase, a 15% increase to 190%increase, a 15% increase to 180% increase, a 15% increase to 170%increase, a 15% increase to 160% increase, a 15% increase to 150%increase, a 15% increase to 140% increase, a 15% increase to 130%increase, a 15% increase to 120% increase, a 15% increase to 110%increase, a 15% increase to 100% increase, a 15% increase to 90%increase, a 15% increase to 80% increase, a 15% increase to 70%increase, a 15% increase to 60% increase, a 15% increase to 50%increase, a 15% increase to 40% increase, a 15% increase to 30%increase, a 15% increase to 25% increase, a 15% increase to 20%increase, a 20% increase to 500% increase, a 20% increase to 450%increase, a 20% increase to 400% increase, a 20% increase to 350%increase, a 20% increase to 300% increase, a 20% increase to 250%increase, a 20% increase to 200% increase, a 20% increase to 190%increase, a 20% increase to 180% increase, a 20% increase to 170%increase, a 20% increase to 160% increase, a 20% increase to 150%increase, a 20% increase to 140% increase, a 20% increase to 130%increase, a 20% increase to 120% increase, a 20% increase to 110%increase, a 20% increase to 100% increase, a 20% increase to 90%increase, a 20% increase to 80% increase, a 20% increase to 70%increase, a 20% increase to 60% increase, a 20% increase to 50%increase, a 20% increase to 40% increase, a 20% increase to 30%increase, a 20% increase to 25% increase, a 25% increase to 500%increase, a 25% increase to 450% increase, a 25% increase to 400%increase, a 25% increase to 350% increase, a 25% increase to 300%increase, a 25% increase to 250% increase, a 25% increase to 200%increase, a 25% increase to 190% increase, a 25% increase to 180%increase, a 25% increase to 170% increase, a 25% increase to 160%increase, a 25% increase to 150% increase, a 25% increase to 140%increase, a 25% increase to 130% increase, a 25% increase to 120%increase, a 25% increase to 110% increase, a 25% increase to 100%increase, a 25% increase to 90% increase, a 25% increase to 80%increase, a 25% increase to 70% increase, a 25% increase to 60%increase, a 25% increase to 50% increase, a 25% increase to 40%increase, a 25% increase to 30% increase, a 30% increase to 500%increase, a 30% increase to 450% increase, a 30% increase to 400%increase, a 30% increase to 350% increase, a 30% increase to 300%increase, a 30% increase to 250% increase, a 30% increase to 200%increase, a 30% increase to 190% increase, a 30% increase to 180%increase, a 30% increase to 170% increase, a 30% increase to 160%increase, a 30% increase to 150% increase, a 30% increase to 140%increase, a 30% increase to 130% increase, a 30% increase to 120%increase, a 30% increase to 110% increase, a 30% increase to 100%increase, a 30% increase to 90% increase, a 30% increase to 80%increase, a 30% increase to 70% increase, a 30% increase to 60%increase, a 30% increase to 50% increase, a 30% increase to 40%increase, a 40% increase to 500% increase, a 40% increase to 450%increase, a 40% increase to 400% increase, a 40% increase to 350%increase, a 40% increase to 300% increase, a 40% increase to 250%increase, a 40% increase to 200% increase, a 40% increase to 190%increase, a 40% increase to 180% increase, a 40% increase to 170%increase, a 40% increase to 160% increase, a 40% increase to 150%increase, a 40% increase to 140% increase, a 40% increase to 130%increase, a 40% increase to 120% increase, a 40% increase to 110%increase, a 40% increase to 100% increase, a 40% increase to 90%increase, a 40% increase to 80% increase, a 40% increase to 70%increase, a 40% increase to 60% increase, a 40% increase to 50%increase, a 50% increase to 500% increase, a 50% increase to 450%increase, a 50% increase to 400% increase, a 50% increase to 350%increase, a 50% increase to 300% increase, a 50% increase to 250%increase, a 50% increase to 200% increase, a 50% increase to 190%increase, a 50% increase to 180% increase, a 50% increase to 170%increase, a 50% increase to 160% increase, a 50% increase to 150%increase, a 50% increase to 140% increase, a 50% increase to 130%increase, a 50% increase to 120% increase, a 50% increase to 110%increase, a 50% increase to 100% increase, a 50% increase to 90%increase, a 50% increase to 80% increase, a 50% increase to 70%increase, a 50% increase to 60% increase, a 60% increase to 500%increase, a 60% increase to 450% increase, a 60% increase to 400%increase, a 60% increase to 350% increase, a 60% increase to 300%increase, a 60% increase to 250% increase, a 60% increase to 200%increase, a 60% increase to 190% increase, a 60% increase to 180%increase, a 60% increase to 170% increase, a 60% increase to 160%increase, a 60% increase to 150% increase, a 60% increase to 140%increase, a 60% increase to 130% increase, a 60% increase to 120%increase, a 60% increase to 110% increase, a 60% increase to 100%increase, a 60% increase to 90% increase, a 60% increase to 80%increase, a 60% increase to 70% increase, a 70% increase to 500%increase, a 70% increase to 450% increase, a 70% increase to 400%increase, a 70% increase to 350% increase, a 70% increase to 300%increase, a 70% increase to 250% increase, a 70% increase to 200%increase, a 70% increase to 190% increase, a 70% increase to 180%increase, a 70% increase to 170% increase, a 70% increase to 160%increase, a 70% increase to 150% increase, a 70% increase to 140%increase, a 70% increase to 130% increase, a 70% increase to 120%increase, a 70% increase to 110% increase, a 70% increase to 100%increase, a 70% increase to 90% increase, a 70% increase to 80%increase, a 80% increase to 500% increase, a 80% increase to 450%increase, a 80% increase to 400% increase, a 80% increase to 350%increase, a 80% increase to 300% increase, a 80% increase to 250%increase, a 80% increase to 200% increase, a 80% increase to 190%increase, a 80% increase to 180% increase, a 80% increase to 170%increase, a 80% increase to 160% increase, a 80% increase to 150%increase, a 80% increase to 140% increase, a 80% increase to 130%increase, a 80% increase to 120% increase, a 80% increase to 110%increase, a 80% increase to 100% increase, a 80% increase to 90%increase, a 90% increase to 500% increase, a 90% increase to 450%increase, a 90% increase to 400% increase, a 90% increase to 350%increase, a 90% increase to 300% increase, a 90% increase to 250%increase, a 90% increase to 200% increase, a 90% increase to 190%increase, a 90% increase to 180% increase, a 90% increase to 170%increase, a 90% increase to 160% increase, a 90% increase to 150%increase, a 90% increase to 140% increase, a 90% increase to 130%increase, a 90% increase to 120% increase, a 90% increase to 110%increase, a 90% increase to 100% increase, a 100% increase to 500%increase, a 100% increase to 450% increase, a 100% increase to 400%increase, a 100% increase to 350% increase, a 100% increase to 300%increase, a 100% increase to 250% increase, a 100% increase to 200%increase, a 100% increase to 190% increase, a 100% increase to 180%increase, a 100% increase to 170% increase, a 100% increase to 160%increase, a 100% increase to 150% increase, a 100% increase to 140%increase, a 100% increase to 130% increase, a 100% increase to 120%increase, a 100% increase to 110% increase, a 110% increase to 500%increase, a 110% increase to 450% increase, a 110% increase to 400%increase, a 110% increase to 350% increase, a 110% increase to 300%increase, a 110% increase to 250% increase, a 110% increase to 200%increase, a 110% increase to 190% increase, a 110% increase to 180%increase, a 110% increase to 170% increase, a 110% increase to 160%increase, a 110% increase to 150% increase, a 110% increase to 140%increase, a 110% increase to 130% increase, a 110% increase to 120%increase, a 120% increase to 500% increase, a 120% increase to 450%increase, a 120% increase to 400% increase, a 120% increase to 350%increase, a 120% increase to 300% increase, a 120% increase to 250%increase, a 120% increase to 200% increase, a 120% increase to 190%increase, a 120% increase to 180% increase, a 120% increase to 170%increase, a 120% increase to 160% increase, a 120% increase to 150%increase, a 120% increase to 140% increase, a 120% increase to 130%increase, a 130% increase to 500% increase, a 130% increase to 450%increase, a 130% increase to 400% increase, a 130% increase to 350%increase, a 130% increase to 300% increase, a 130% increase to 250%increase, a 130% increase to 200% increase, a 130% increase to 190%increase, a 130% increase to 180% increase, a 130% increase to 170%increase, a 130% increase to 160% increase, a 130% increase to 150%increase, a 130% increase to 140% increase, a 140% increase to 500%increase, a 140% increase to 450% increase, a 140% increase to 400%increase, a 140% increase to 350% increase, a 140% increase to 300%increase, a 140% increase to 250% increase, a 140% increase to 200%increase, a 140% increase to 190% increase, a 140% increase to 180%increase, a 140% increase to 170% increase, a 140% increase to 160%increase, a 140% increase to 150% increase, a 150% increase to 500%increase, a 150% increase to 450% increase, a 150% increase to 400%increase, a 150% increase to 350% increase, a 150% increase to 300%increase, a 150% increase to 250% increase, a 150% increase to 200%increase, a 150% increase to 190% increase, a 150% increase to 180%increase, a 150% increase to 170% increase, a 150% increase to 160%increase, a 160% increase to 500% increase, a 160% increase to 450%increase, a 160% increase to 400% increase, a 160% increase to 350%increase, a 160% increase to 300% increase, a 160% increase to 250%increase, a 160% increase to 200% increase, a 160% increase to 190%increase, a 160% increase to 180% increase, a 160% increase to 170%increase, a 170% increase to 500% increase, a 170% increase to 450%increase, a 170% increase to 400% increase, a 170% increase to 350%increase, a 170% increase to 300% increase, a 170% increase to 250%increase, a 170% increase to 200% increase, a 170% increase to 190%increase, a 170% increase to 180% increase, a 180% increase to 500%increase, a 180% increase to 450% increase, a 180% increase to 400%increase, a 180% increase to 350% increase, a 180% increase to 300%increase, a 180% increase to 250% increase, a 180% increase to 200%increase, a 180% increase to 190% increase, a 190% increase to 500%increase, a 190% increase to 450% increase, a 190% increase to 400%increase, a 190% increase to 350% increase, a 190% increase to 300%increase, a 190% increase to 250% increase, a 190% increase to 200%increase, a 200% increase to 500% increase, a 200% increase to 450%increase, a 200% increase to 400% increase, a 200% increase to 350%increase, a 200% increase to 300% increase, a 200% increase to 250%increase, a 250% increase to 500% increase, a 250% increase to 450%increase, a 250% increase to 400% increase, a 250% increase to 350%increase, a 250% increase to 300% increase, a 300% increase to 500%increase, a 300% increase to 450% increase, a 300% increase to 400%increase, a 300% increase to 350% increase, a 350% increase to 500%increase, a 350% increase to 450% increase, a 350% increase to 400%increase, a 400% increase to 500% increase, a 400% increase to 450%increase, or a 450% increase to 500% increase) in one or more (e.g.,two, three, four, five, six, seven, eight, nine, or ten) of: the plasma,serum, or blood level of IL-6; the plasma, serum, or blood level ofIL-2; the plasma, serum, or blood level of IL-1β; the plasma, serum, orblood level of TNFα; the plasma, serum, or blood level of IL-17A; theplasma, serum, or blood level of IL-22; the plasma, serum, or bloodlevel of interferon-γ; the level of blood Th memory cells(CD44⁺CD45RB⁻CD4⁺ cells); and the level of α4β7 expression in bloodcells; e.g., each as compared to the corresponding level in a subjectsystemically administered the same dose of the same TLR agonist. Methodsfor determining the plasma, serum, or blood level of IL-6; the plasma,serum, or blood level of IL-2; the plasma, serum, or blood level ofIL-1β; the plasma, serum, or blood level of TNFα; the plasma, serum, orblood level of IL-17A; the plasma, serum, or blood level of IL-22; theplasma, serum, or blood level of interferon-γ; the level of blood Thmemory cells (CD44⁺CD45RB⁻CD4⁺ cells); and the level of α4β7 expressionin blood cells are known in the art.

In some examples of any of the methods described herein can result,e.g., in a 1% to 99% decrease (or any of the subranges of this rangedescribed herein) in one or more (e.g., two, three, four, five, six, orseven) of: the level of interferon-γ in GI tissue or GI content; thelevel of IL-1β in GI tissue or GI content; the level of IL-6 in GItissue or GI content; the level of IL-22 in GI tissue or GI content; thelevel of IL-17A in GI tissue or GI content; the level of TNFα in GItissue or GI content; and the level of IL-2 in GI tissue or GI content,e.g., as compared to the corresponding level in a subject notadministered a treatment, or not administered a TLR agonist locally asdisclosed herein. Accordingly, in some embodiments, the methodsdescribed herein can result, e.g., in a 1% to 99% decrease (or any ofthe subranges of this range described herein) in one or more (e.g., two,three, four, five, six, or seven) of the level of interferon-γ; thelevel of IL-1β; the level of IL-6; the level of IL-22; the level ofIL-17A; the level of TNFα; and the level of IL-2, in the duodenum tissueproximate to one or more sites of disease. Accordingly, in someembodiments, the methods described herein can result, e.g., in a 1% to99% decrease (or any of the subranges of this range described herein) inone or more (e.g., two, three, four, five, six, or seven) of the levelof interferon-γ; the level of IL-1β; the level of IL-6; the level ofIL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, inthe ileum tissue proximate to one or more sites of disease. Accordingly,in some embodiments, the methods described herein can result, e.g., in a1% to 99% decrease (or any of the subranges of this range describedherein) in one or more (e.g., two, three, four, five, six, or seven) ofthe level of interferon-γ; the level of IL-1⊕; the level of IL-6; thelevel of IL-22; the level of IL-17A; the level of TNFα; and the level ofIL-2, in the jejunum tissue proximate to one or more sites of disease.Accordingly, in some embodiments, the methods described herein canresult, e.g., in a 1% to 99% decrease (or any of the subranges of thisrange described herein) in one or more (e.g., two, three, four, five,six, or seven) of the level of interferon-γ; the level of IL-1β; thelevel of IL-6; the level of IL-22; the level of IL-17A; the level ofTNFα; and the level of IL-2, in the cecum tissue proximate to one ormore sites of disease. Accordingly, in some embodiments, the methodsdescribed herein can result, e.g., in a 1% to 99% decrease (or any ofthe subranges of this range described herein) in one or more (e.g., two,three, four, five, six, or seven) of the level of interferon-γ; thelevel of IL-1β; the level of IL-6; the level of IL-22; the level ofIL-17A; the level of TNFα; and the level of IL-2, in the ascending colontissue proximate to one or more sites of disease. Accordingly, in someembodiments, the methods described herein can result, e.g., in a 1% to99% decrease (or any of the subranges of this range described herein) inone or more (e.g., two, three, four, five, six, or seven) of the levelof interferon-γ; the level of IL-1β; the level of IL-6; the level ofIL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, inthe transverse colon tissue proximate to one or more sites of disease.Accordingly, in some embodiments, the methods described herein canresult, e.g., in a 1% to 99% decrease (or any of the subranges of thisrange described herein) in one or more (e.g., two, three, four, five,six, or seven) of the level of interferon-γ; the level of IL-1β; thelevel of IL-6; the level of IL-22; the level of IL-17A; the level ofTNFα; and the level of IL-2, in the descending colon tissue proximate toone or more sites of disease. Accordingly, in some embodiments, themethods described herein can result, e.g., in a 1% to 99% decrease (orany of the subranges of this range described herein) in one or more(e.g., two, three, four, five, six, or seven) of the level ofinterferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22;the level of IL-17A; the level of TNFα; and the level of IL-2, in thesigmoid colon tissue proximate to one or more sites of disease.

In some embodiments, the TLR agonist is delivered to the location by aprocess that does not comprise systemic transport of the TLR agonist.

In some embodiments, the amount of the TLR agonist that is administeredis from about 1 mg to about 500 mg. In some embodiments, the amount ofthe TLR agonist that is administered is from about 1 mg to about 100 mg.In some embodiments, the amount of the TLR agonist that is administeredis from about 5 mg to about 40 mg. In some embodiments, the amount ofcobitolimod that is administered is about 30 mg.

In some embodiments, the amount of the TLR agonist that is administeredis less than an amount that is effective when the TLR agonist isdelivered systemically.

In some embodiments, the amount of the TLR agonist that is administeredis an induction dose. In some embodiments, such induction dose iseffective to induce remission of the TNF and cytokine storm and healingof acute inflammation and lesions. In some embodiments, the inductiondose is administered once a day. In some embodiments, the induction doseis administered once every three days. In some embodiments, theinduction dose is administered once a week. In some embodiments, theinduction dose is administered once a day, once every three days, oronce a week, over a period of about 6-8 weeks.

In some embodiments, the method comprises administering (i) an amount ofthe TLR agonist that is an induction dose, and (ii) an amount of the TLRagonist that is a maintenance dose, in this order. In some embodiments,step (ii) is repeated one or more times. In some embodiments, theinduction dose is equal to the maintenance dose. In some embodiments,the induction dose is greater than the maintenance dose. In someembodiments, the induction dose is five times greater than themaintenance dose. In some embodiments, the induction dose is two timesgreater than the maintenance dose.

In some embodiments, the induction dose is the same as or higher than aninduction dose administered systemically for treatment of the samedisorder to a subject. In more particular embodiments, the inductiondose is the same as or higher than an induction dose administeredsystemically for treatment of the same disorder to a subject, and themaintenance dose is lower than the maintenance dose administeredsystemically for treatment of the same disorder to a subject. In someembodiments, the induction dose is the same as or higher than aninduction dose administered systemically for treatment of the samedisorder to a subject, and the maintenance dose is higher than themaintenance dose administered systemically for treatment of the samedisorder to a subject.

In some embodiments an induction dose of TLR agonist and a maintenancedose of TLR agonist are each administered to the subject byadministering a pharmaceutical composition comprising a therapeuticallyeffective amount of the TLR agonist, wherein the pharmaceuticalcomposition is a device. In some embodiments an induction dose of TLRagonist is administered to the subject in a different manner from themaintenance dose. As an example, the induction dose may be administeredsystemically. In some embodiments, the induction dose may beadministered other than orally. As an example, the induction dose may beadministered rectally. As an example, the induction dose may beadministered intravenously. As an example, the induction dose may beadministered subcutaneously. In some embodiments, the induction dose maybe administered by spray catheter.

In some embodiments, the concentration of the TLR agonist delivered atthe location in the gastrointestinal tract is 10%, 25%, 50%, 75%, 100%,200%, 300%, 400%, 500%, 1000%, 2000% greater than the concentration ofTLR agonist in plasma.

In some embodiments, the method provides a concentration of the TLRagonist at a location that is a site of disease or proximate to a siteof disease that is 2-100 times greater than at a location that is not asite of disease or proximate to a site of disease.

In some embodiments, the method comprises delivering the TLR agonist atthe location in the gastrointestinal tract as a single bolus.

In some embodiments, the method comprises delivering the TLR agonist atthe location in the gastrointestinal tract as more than one bolus.

In some embodiments, the method comprises delivering the TLR agonist atthe location in the gastrointestinal tract in a continuous manner.

In some embodiments, the method comprises delivering the TLR agonist atthe location in the gastrointestinal tract over a time period of 20 ormore minutes.

In some embodiments, the method provides a concentration of the TLRagonist in the plasma of the subject that is less than 10 μg/ml. In someembodiments, the method provides a concentration of the TLR agonist inthe plasma of the subject that is less than 3 μg/ml. In someembodiments, the method provides a concentration of the TLR agonist inthe plasma of the subject that is less than 1 μg/ml. In someembodiments, the method provides a concentration of the TLR agonist inthe plasma of the subject that is less than 0.3 μg/ml. In someembodiments, the method provides a concentration of the TLR agonist inthe plasma of the subject that is less than 0.1 μg/ml. In someembodiments, the method provides a concentration of the TLR agonist inthe plasma of the subject that is less than 0.01 μg/ml. In someembodiments, the values of the concentration of the TLR agonist in theplasma of the subject provided herein refer to C_(trough), that is, thelowest value of the concentration prior to administration of the nextdose.

In some embodiments, the method provides a concentration C_(max) of theTLR agonist in the plasma of the subject that is less than 10 μg/ml. Insome embodiments, the method provides a concentration C_(max) of the TLRagonist in the plasma of the subject that is less than 3 μg/ml. In someembodiments, the method provides a concentration C_(max) of the TLRagonist in the plasma of the subject that is less than 1 μg/ml. In someembodiments, the method provides a concentration C_(max) of the TLRagonist in the plasma of the subject that is less than 0.3 μg/ml. Insome embodiments, the method provides a concentration C_(max) of the TLRagonist in the plasma of the subject that is less than 0.1 μg/ml. Insome embodiments, the method provides a concentration C_(max) of the TLRagonist in the plasma of the subject that is less than 0.01 μg/ml.

In some embodiments, the method does not comprise delivering a TLRagonist rectally to the subject.

In some embodiments, the method does not comprise delivering a TLRagonist via an enema to the subject.

In some embodiments, the method does not comprise delivering a TLRagonist via suppository to the subject.

In some embodiments, the method does not comprise delivering a TLRagonist via instillation to the rectum of a subject.

In some embodiments, the methods disclosed herein comprise producing atherapeutically effective degradation product of the TLR agonist in thegastrointestinal tract. In some embodiments, the degradation product isa therapeutic antibody fragment. In some embodiments, a therapeuticallyeffective amount of the degradation product is produced.

In some embodiments, the antibody can be a humanized antibody, achimeric antibody, a multivalent antibody, or a fragment thereof. Insome embodiments, an antibody can be a scFv-Fc (Sokolowska-Wedzina etal., Mol. Cancer Res. 15(8):1040-1050, 2017), a VHH domain (Li et al.,Immunol. Lett. 188:89-95, 2017), a VNAR domain (Hasler et al., Mol.Immunol. 75:28-37, 2016), a (scFv)₂, a minibody (Kim et al., PLoS One10(1):e113442, 2014), or a BiTE. In some embodiments, an antibody can bea DVD-Ig (Wu et al., Nat. Biotechnol. 25(11):1290-1297, 2007; WO08/024188; WO 07/024715), and a dual-affinity re-targeting antibody(DART) (Tsai et al., Mol. Ther. Oncolytics 3:15024, 2016), a triomab(Chelius et al., MAbs 2(3):309-319, 2010), kih IgG with a common LC(Kontermann et al., Drug Discovery Today 20(7):838-847, 2015), acrossmab (Regula et al., EMBO Mol. Med. 9(7):985, 2017), an ortho-FabIgG (Kontermann et al., Drug Discovery Today 20(7):838-847, 2015), a2-in-1-IgG (Kontermann et al., Drug Discovery Today 20(7):838-847,2015), IgG-scFv (Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014),scFv2-Fc (Natsume et al., J. Biochem. 140(3):359-368, 2006), abi-nanobody (Kontermann et al., Drug Discovery Today 20(7):838-847,2015), tanden antibody (Kontermann et al., Drug Discovery Today20(7):838-847, 2015), a DART-Fc (Kontermann et al., Drug Discovery Today20(7):838-847, 2015), a scFv-HSA-scFv (Kontermann et al., Drug DiscoveryToday 20(7):838-847, 2015), DNL-Fab3 (Kontermann et al., Drug DiscoveryToday 20(7):838-847, 2015), DAF (two-in-one or four-in-one), DutaMab,DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pairantibody, Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, Fcab,kλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv,scFv-(L)-IgG, IgG (L,H)-Fc, IgG(H)-V, V(H)—IgG, IgG(L)-V, V(L)-IgG, KIHIgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody(e.g., antibodies derived from Camelus bactriamus, Calelus dromaderius,or Lama paccos) (U.S. Pat. No. 5,759,808; Stijlemans et al., J. Biol.Chem. 279:1256-1261, 2004; Dumoulin et al., Nature 424:783-788, 2003;and Pleschberger et al., Bioconjugate Chem. 14:440-448, 2003),nanobody-HSA, a diabody (e.g., Poljak, Structure 2(12):1121-1123, 1994;Hudson et al., J. Immunol. Methods 23(1-2):177-189, 1999), a TandAb(Reusch et al., mAbs 6(3):727-738, 2014), scDiabody (Cuesta et al.,Trends in Biotechnol. 28(7):355-362, 2010), scDiabody-CH3 (Sanz et al.,Trends in Immunol. 25(2):85-91, 2004), Diabody-CH3 (Guo et al., TripleBody, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv,scFv-CH-CL-scFv, F(ab′)2-scFV2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb,scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody (Huston et al.,Human Antibodies 10(3-4):127-142, 2001; Wheeler et al., Mol. Ther.8(3):355-366, 2003; Stocks, Drug Discov. Today 9(22):960-966, 2004),dock and lock bispecific antibody, ImmTAC, HSAbody, scDiabody-HSA,tandem scFv, IgG-IgG, Cov-X-Body, and scFv1-PEG-scFv2.

Non-limiting examples of an antigen-binding fragment of an antibodyinclude an Fv fragment, a Fab fragment, a F(ab′)2 fragment, and a Fab′fragment. Additional examples of an antigen-binding fragment of anantibody is an antigen-binding fragment of an IgG (e.g., anantigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., anantigen-binding fragment of a human or humanized IgG, e.g., human orhumanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of anIgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., anantigen-binding fragment of a human or humanized IgA, e.g., a human orhumanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., anantigen-binding fragment of a human or humanized IgD); anantigen-binding fragment of an IgE (e.g., an antigen-binding fragment ofa human or humanized IgE); or an antigen-binding fragment of an IgM(e.g., an antigen-binding fragment of a human or humanized IgM).

In some embodiments, an antibody can be an IgNAR, a bispecific antibody(Milstein and Cuello, Nature 305:537-539, 1983; Suresh et al., Methodsin Enzymology 121:210, 1986; WO 96/27011; Brennan et al., Science229:81, 1985; Shalaby et al., J. Exp. Med. 175:217-225, 1992; Kolstelnyet al., J. Immunol. 148(5):1547-1553, 1992; Hollinger et al., Proc.Natl. Acad. Sci. U.S.A. 90:6444-6448, 1993; Gruber et al., J. Immunol.152:5368, 1994; Tuft et al., J. Immunol. 147:60, 1991), a bispecificdiabody, a triabody (Schoonooghe et al., BMC Biotechnol. 9:70, 2009), atetrabody, scFv-Fc knobs-into-holes, a scFv-Fc-scFv, a (Fab′scFv)₂, aV-IgG, a IvG-V, a dual V domain IgG, a heavy chain immunoglobulin or acamelid (Holt et al., Trends Biotechnol. 21(11):484-490, 2003), anintrabody, a monoclonal antibody (e.g., a human or humanized monoclonalantibody), a heteroconjugate antibody (e.g., U.S. Pat. No. 4,676,980), alinear antibody (Zapata et al., Protein Eng. 8(10:1057-1062, 1995), atrispecific antibody (Tutt et al., J. Immunol. 147:60, 1991), aFabs-in-Tandem immunoglobulin (WO 15/103072), or a humanized camelidantibody.

In some embodiments, the methods comprising administering the TLRagonist in the manner disclosed herein disclosed herein result in areduced immunosuppressive properties relative to methods ofadministration of the TLR agonist systemically.

In some embodiments, the methods comprising administering the TLRagonist in the manner disclosed herein disclosed herein result inreduced immunogenicity relative to methods of administration of the TLRagonist systemically.

Methods for Treating Colitis in Subjects in Immune-Oncology Therapy

In some embodiments, provided herein is a method for treating colitis asdisclosed herein in a subject, comprising releasing a TLR agonist at alocation in the gastrointestinal tract of the subject that is proximateto one or more sites of disease, wherein the method comprisesadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of the TLR agonist, wherein the colitisis associated with treatment of the subject with one or moreimmuno-oncology agents. In some embodiments, the pharmaceuticalcomposition is an ingestible device. In some embodiments, thepharmaceutical composition is an ingestible device and the methodcomprises administering orally to the subject the pharmaceuticalcomposition.

In some embodiments, at least one of the one or more immuno-oncologyagents is a chemotherapeutic agent. In some embodiments, thechemotherapeutic agent is a chemotherapeutic immunomodulator. In someembodiments, the chemotherapeutic immunomodulator is an immunecheckpoint inhibitor.

In some embodiments, the immune checkpoint inhibitor targets an immunecheckpoint protein or decreases an activity of an immune checkpointprotein selected from the group of CTLA-4, PD-1, PD-L1, PD-1-PD-L1,PD-1-PD-L2, interleukin 2 (IL 2), indoleamine 2,3-dioxygenase (IDO), IL10, transforming growth factor-β (TGFβ), T cell immunoglobulin and mucin3 (TIM3 or HAVCR2), Galectin 9-TIM3, Phosphatidylserine-TIM3, lymphocyteactivation gene 3 protein (LAG3), MHC class II-LAG3, 4 1BB-4 1BB ligand,OX40-OX40 ligand, GITR, GITR ligand-GITR, CD27, CD70-CD27, TNFRSF25,TNFRSF25-TL1A, CD40L, CD40-CD40 ligand, HVEM-LIGHT-LTA, HVEM, HVEM-BTLA,HVEM-CD160, HVEM-LIGHT, HVEM-BTLA-CD160, CD80, CD80-PDL-1, PDL2-CD80,CD244, CD48-CD244, CD244, ICOS, ICOS-ICOS ligand, B7 H3, B7 H4, VISTA,TMIGD2, HHLA2-TMIGD2, Butyrophilins, including BTNL2, Siglec family,TIGIT and PVR family members, KIRs, ILTs and LIRs, NKG2D and NKG2A, MICAand MICB, CD244, CD28, CD86-CD28, CD86-CTLA, CD80-CD28, CD39, CD73Adenosine-CD39-CD73, CXCR4-CXCL12, Phosphatidylserine, TIM3,Phosphatidylserine-TIM3, SIRPA-CD47, VEGF, Neuropilin, CD160, CD30, andCD155.

In some examples, the immune checkpoint inhibitor is selected from thegroup consisting of: Urelumab, PF 05082566, MEDI6469, TRX518,Varlilumab, CP 870893, Pembrolizumab (PD1), Nivolumab (PD1),Atezolizumab (formerly MPDL3280A) (PDL1), MEDI4736 (PD-L1), Avelumab(PD-L1), PDR001 (PD1), BMS 986016, MGA271, Lirilumab, IPH2201,Emactuzumab, INCB024360, Galunisertib, Ulocuplumab, BKT140, Bavituximab,CC 90002, Bevacizumab, and MNRP1685A, and MGA271.

In some examples, the immune checkpoint inhibitor targets or decreasesan activity of CTLA-4. In some embodiments, the immune checkpointinhibitor is an antibody. In some embodiments, the antibody isipilimumab or tremelimumab.

In some examples, the immune checkpoint inhibitor targets PD1 or PD-L1.In some examples, the immune checkpoint inhibitor is selected fromnivolumab, lambroizumab, and BMS-936559.

In some embodiments, at least one of the one or more immuno-oncologyagents is a T-cell capable of expressing a chimeric antigen receptor(CAR). In some embodiments, at least one of the one or moreimmuno-oncology agents is a PI-3-kinase inhibitor.

In some embodiments, the treatment of the subject with one or moreimmuno-oncology agents further comprises treatment of the subject withan immunosuppressant.

In some embodiments, provided herein is a method for reducing thedevelopment of colitis in a subject administered an immuno-oncologyagent, comprising releasing a TLR agonist at a location in thegastrointestinal tract of the subject that is proximate to one or moresites of disease, wherein the method comprises administering to thesubject a pharmaceutical composition comprising a therapeuticallyeffective amount of the TLR agonist. In some embodiments, thepharmaceutical composition is an ingestible device. In some embodiments,the pharmaceutical composition is an ingestible device and the methodcomprises administering orally to the subject the pharmaceuticalcomposition.

In some embodiments of these methods, a subject is administered at leastone dose of an immuno-oncology agent prior to administering apharmaceutical composition comprising any of the devices describedherein as described herein to the subject. In some embodiments of thesemethods, a subject is first administered any of the devices as describedherein, prior to administration of the first dose of the immuno-oncologyagent. In some embodiments of these methods, the immuno-oncology agentis administered at substantially the same time as the device describedherein.

Also provided herein are methods of treating a subject having a cancerthat include: administering a first dose of an immuno-oncology agent tothe subject; monitoring one or more biomarkers, markers, or symptoms ofcolitis (e.g., any of the biomarkers, markers, or symptoms of colitisdescribed herein or known in the art); identifying a subject having alevel of a biomarker or marker, or having a symptom of colitis; andreleasing a TLR agonist at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of disease, whereinthe method comprises administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of the TLRagonist. In some embodiments, the pharmaceutical composition is aningestible device. In some embodiments, the pharmaceutical compositionis an ingestible device and the method comprises administering orally tothe subject the pharmaceutical composition.

Also provided herein are methods of reducing the severity of colitis ina subject having a cancer and administered an immuno-oncology agent thatinclude administering to the subject any of the devices describedherein.

In some embodiments, provided herein is a method for treating colitis ina subject comprising:

determining that the subject has colitis associated with treatment ofthe subject with one or more immuno-oncology agents; and

releasing a TLR agonist at a location in the gastrointestinal tract ofthe subject that is proximate to one or more sites of colitis, whereinthe method comprises administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of the TLRagonist. In some embodiments, the pharmaceutical composition is aningestible device. In some embodiments, the pharmaceutical compositionis an ingestible device and the method comprises administering orally tothe subject the pharmaceutical composition.

In some embodiments, provided herein is a method for treating colitis ina subject comprising:

determining that the subject has colitis associated with treatment ofthe subject with one or more immuno-oncology agents; and

administering to the subject an ingestible device comprising any of theTLR agonists described herein, to treat the colitis.

In some embodiments, provided herein is a method for treating colitis,

comprising releasing a TLR agonist at a location in the gastrointestinaltract of a subject who has been determined to have colitis associatedwith treatment of the subject with one or more immuno-oncology agents,wherein the location is proximate to one or more sites of colitis,wherein the method comprises administering to the subject apharmaceutical composition comprising a therapeutically effective amountof the TLR agonist. In some embodiments, the pharmaceutical compositionis an ingestible device. In some embodiments, the pharmaceuticalcomposition is an ingestible device and the method comprisesadministering orally to the subject the pharmaceutical composition.

In some embodiments, provided herein is a method for treating colitis,comprising administering an ingestible device comprising any of the TLRagonists described herein to a subject who has been determined to havecolitis associated with treatment of the subject with one or moreimmuno-oncology agents.

In some embodiments, provided herein is an ingestible device comprisingany of the TLR agonists described herein for treating colitis associatedwith treatment of a subject with one or more immuno-oncology agents.

Monitoring Progress of Disease

In some embodiments, the methods provided herein comprise monitoring theprogress of the disease. In some embodiments, monitoring the progress ofthe disease comprises measuring the levels of IBD serological markers.In some embodiments, monitoring the progress of the disease comprisesdetermining mucosal healing at the location of release. In someembodiments, monitoring the progress of the disease comprisesdetermining the Crohn's Disease Activity Index (CDAI) over a period ofabout 6-8 weeks, or over a period of about 52 weeks, followingadministration of the TLR agonist. In some embodiments, monitoring theprogress of the disease comprises determining the Harvey-Bradshaw Index(HBI) following administration of the TLR agonist. Possible markers mayinclude the following: anti-glycan antibodies: anti-Saccharomycescerevisiae (ASCA); anti-laminaribioside (ALCA); anti-chitobioside(ACCA); anti-mannobioside (AMCA); anti-laminarin (anti-L); anti-chitin(anti-C) antibodies: anti-outer membrane porin C (anti-OmpC), anti-Cbir1flagellin; anti-12 antibody; autoantibodies targeting the exocrinepancreas (PAB); perinuclear anti-neutrophil antibody (pANCA). In someembodiments, monitoring the progress of the disease comprises measuringTLR agonist levels in serum over a period of about 1-14 weeks, such asabout 6-8 weeks following administration of the TLR agonist, includingat the 6-8 week time point. In some embodiments, monitoring the progressof the disease comprises measuring TLR agonist levels in serum over aperiod of about 52 weeks following administration of the TLR agonist,including at the 52 week time point.

Patients Condition, Diagnosis and Treatment

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises one or more of the following:

-   -   a) identifying a subject having a disease of the        gastrointestinal tract, for example by endoscopy or colonoscopy;    -   b) determination of the severity of the disease, for example        with reference to the Mayo Clinic Score, the Crohn's Disease        Activity Index (CDAI), the Harvey-Bradshaw Index (HBI), or a        combination of the above;    -   c) determination of the location of the disease, for example as        determined by the presence of lesions indicative of the disease;    -   d) evaluating the subject for suitability to treatment, for        example by determining the patency of the subject's GI tract,        for example if the indication is small intestinal diseases,        pancolitis, Crohn's disease, or if the patients has strictures        or fistulae;    -   e) administration of an induction dose or of a maintenance dose        of a drug, such as the TLR agonist or such as another drug that        is effective in the treatment of IBD conditions;    -   f) monitoring the progress of the disease, for example with        reference to the Mayo Clinic Score, the Crohn's Disease Activity        Index (CDAI), the Harvey-Bradshaw Index (HBI), the PRO, PRO2 or        PRO3 tools, or a combination of the above; and/or    -   g) optionally repeating steps e) and f) one or more times, for        example over a period of about 1-14 weeks, such as about 6-8        weeks following administration of the TLR agonist, including at        the 6-8 week time point, or over a period of about 52 weeks        following administration of the TLR agonist, including at the 52        week time point.

As used herein, an induction dose is a dose of drug that may beadministered, for example, at the beginning of a course of treatment,and that is higher than the maintenance dose administered duringtreatment. An induction dose may also be administered during treatment,for example if the condition of the patients becomes worse.

As used herein, a maintenance dose is a dose of drug that is provided ona repetitive basis, for example at regular dosing intervals.

In some embodiments the TLR agonist is released from an ingestibledevice.

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises a) hereinabove.

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises b) hereinabove.

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises c) hereinabove.

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises d) hereinabove.

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises e) hereinabove.

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises f) hereinabove.

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises g) hereinabove.

In some embodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises a) and b) hereinabove. In some embodimentsherein, the method of treating a disease of the gastrointestinal tractthat comprises releasing a TLR agonist at a location in thegastrointestinal tract that is proximate to one or more sites of diseasecomprises a) and c) hereinabove. In some embodiments herein, the methodof treating a disease of the gastrointestinal tract that comprisesreleasing a TLR agonist at a location in the gastrointestinal tract thatis proximate to one or more sites of disease comprises a) and d)hereinabove. In some embodiments herein, the method of treating adisease of the gastrointestinal tract that comprises releasing a TLRagonist at a location in the gastrointestinal tract that is proximate toone or more sites of disease comprises a) and e) hereinabove. In someembodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises a) and f) hereinabove. In some embodimentsherein, the method of treating a disease of the gastrointestinal tractthat comprises releasing a TLR agonist at a location in thegastrointestinal tract that is proximate to one or more sites of diseasecomprises a) and g) hereinabove. In some embodiments herein, the methodof treating a disease of the gastrointestinal tract that comprisesreleasing a TLR agonist at a location in the gastrointestinal tract thatis proximate to one or more sites of disease comprises b) and c)hereinabove. In some embodiments herein, the method of treating adisease of the gastrointestinal tract that comprises releasing a TLRagonist at a location in the gastrointestinal tract that is proximate toone or more sites of disease comprises b) and d) hereinabove. In someembodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises b) and e) hereinabove. In some embodimentsherein, the method of treating a disease of the gastrointestinal tractthat comprises releasing a TLR agonist at a location in thegastrointestinal tract that is proximate to one or more sites of diseasecomprises b) and f) hereinabove. In some embodiments herein, the methodof treating a disease of the gastrointestinal tract that comprisesreleasing a TLR agonist at a location in the gastrointestinal tract thatis proximate to one or more sites of disease comprises b) and g)hereinabove. In some embodiments herein, the method of treating adisease of the gastrointestinal tract that comprises releasing a TLRagonist at a location in the gastrointestinal tract that is proximate toone or more sites of disease comprises c) and d) hereinabove. In someembodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises c) and e) hereinabove. In some embodimentsherein, the method of treating a disease of the gastrointestinal tractthat comprises releasing a TLR agonist at a location in thegastrointestinal tract that is proximate to one or more sites of diseasecomprises c) and f) hereinabove. In some embodiments herein, the methodof treating a disease of the gastrointestinal tract that comprisesreleasing a TLR agonist at a location in the gastrointestinal tract thatis proximate to one or more sites of disease comprises c) and g)hereinabove. In some embodiments herein, the method of treating adisease of the gastrointestinal tract that comprises releasing a TLRagonist at a location in the gastrointestinal tract that is proximate toone or more sites of disease comprises d) and e) hereinabove. In someembodiments herein, the method of treating a disease of thegastrointestinal tract that comprises releasing a TLR agonist at alocation in the gastrointestinal tract that is proximate to one or moresites of disease comprises d) and f) hereinabove. In some embodimentsherein, the method of treating a disease of the gastrointestinal tractthat comprises releasing a TLR agonist at a location in thegastrointestinal tract that is proximate to one or more sites of diseasecomprises d) and g) hereinabove. In some embodiments herein, the methodof treating a disease of the gastrointestinal tract that comprisesreleasing a TLR agonist at a location in the gastrointestinal tract thatis proximate to one or more sites of disease comprises e) and f)hereinabove. In some embodiments herein, the method of treating adisease of the gastrointestinal tract that comprises releasing a TLRagonist at a location in the gastrointestinal tract that is proximate toone or more sites of disease comprises g) hereinabove.

In some embodiments, one or more steps a) to e) herein compriseendoscopy of the gastrointestinal tract. In some embodiments, one ormore steps a) to e) herein comprise colonoscopy of the gastrointestinaltract. In some embodiments, one or more steps a) to e) herein isperformed one or more times. In some embodiments, such one or more ofsuch one or more steps a) to e) is performed after releasing the TLRagonist at the location in the gastrointestinal tract that is proximateto one or more sites of disease.

In some embodiments, the method comprises administering one or moremaintenance doses following administration of the induction dose in stepe). In some embodiments an induction dose of TLR agonist and amaintenance dose of TLR agonist are each administered to the subject byadministering a pharmaceutical composition comprising a therapeuticallyeffective amount of the TLR agonist. In some embodiments an inductiondose of TLR agonist is administered to the subject in a different mannerfrom the maintenance dose. As an example, the maintenance dose may beadministered systemically, while the maintenance dose is administeredlocally using a device. In one embodiment, a maintenance dose isadministered systemically, and an induction dose is administered using adevice every 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 35, 40, or 45days. In another embodiment, a maintenance dose is administeredsystemically, and an induction dose is administered when a disease flareup is detected or suspected.

In some embodiments, the induction dose is a dose of the TLR agonistadministered in an ingestible device as disclosed herein. In someembodiments, the maintenance dose is a dose of the TLR agonistadministered in an ingestible device as disclosed herein.

In some embodiments, the induction dose is a dose of the TLR agonistadministered in an ingestible device as disclosed herein. In someembodiments, the maintenance dose is a dose of the TLR agonist deliveredsystemically, such as orally with a tablet or capsule, orsubcutaneously, or intravenously.

In some embodiments, the induction dose is a dose of the TLR agonistdelivered systemically, such as orally with a tablet or capsule, orsubcutaneously, or intravenously. In some embodiments, the maintenancedose is a dose of the TLR agonist administered in an ingestible deviceas disclosed herein.

In some embodiments, the induction dose is a dose of the TLR agonistadministered in an ingestible device as disclosed herein. In someembodiments, the maintenance dose is a dose of a second agent asdisclosed herein delivered systemically, such as orally with a tablet orcapsule, or subcutaneously, or intravenously.

In some embodiments, the induction dose is a dose of a second agent asdisclosed herein delivered systemically, such as orally with a tablet orcapsule, or subcutaneously, or intravenously. In some embodiments, themaintenance dose is a dose of the TLR agonist administered in aningestible device as disclosed herein.

In one embodiment of the methods provided herein, the patient is notpreviously treated with a TLR agonist. In one embodiment, thegastrointestinal inflammatory disorder is an inflammatory bowel disease.In one embodiment, the inflammatory bowel disease is ulcerative colitisor Crohn's disease. In one embodiment, the inflammatory bowel disease isulcerative colitis and the response is selected from clinical response,mucosal healing and remission. In certain embodiments, remission in thepatient is determined to be induced when the Mayo Clinic Score <2 and noindividual subscore >1, which is also referred to as clinical remission.In certain embodiments, mucosal healing is determined to have occurredwhen the patient is determined to have an endoscopy subscore of 0 or 1as assessed by flexible sigmoidoscopy. In certain such embodiments,patients who experience mucosal healing are determined to have anendoscopy subscore of 0. In certain embodiments, clinical response isdetermined to have occurred when the patient experiences a 3-pointdecrease and 30% reduction from baseline in MCS and >1-point decrease inrectal bleeding subscore or absolute rectal bleeding score of 0 or 1.

In some embodiments, the method comprises identifying the disease sitesubstantially at the same time as releasing the TLR agonist.

In some embodiments, the method comprises monitoring the progress of thedisease. In some embodiments, monitoring the progress of the diseasecomprises measuring the weight of the subject over a period of about1-14 weeks, such as about 6-8 weeks following administration of the TLRagonist, including at the 6-8 week time point, or over a period of about52 weeks following administration of the TLR agonist, including at the52 week time point. In some embodiments, monitoring the progress of thedisease comprises measuring the food intake of the subject; measuringthe level of blood in the feces of the subject; measuring the level ofabdominal pain of the subject; and/or a combination of the above, forexample over a period of about 1-14 weeks, such as about 6-8 weeksfollowing administration of the TLR agonist, including at the 6-8 weektime point, or over a period of about 52 weeks following administrationof the TLR agonist, including at the 52 week time point.

In some embodiments, the method comprises administering a TLR agonistwith a spray catheter. For example, administering a TLR agonist with aspray catheter may be performed in step (e) hereinabove.

In some embodiments, the method does not comprise administering a TLRagonist with a spray catheter.

In some embodiments, data obtained from cell culture assays and animalstudies can be used in formulating an appropriate dosage of any givenTLR agonist. The effectiveness and dosing of any TLR agonist can bedetermined by a health care professional or veterinary professionalusing methods known in the art, as well as by the observation of one ormore disease symptoms in a subject (e.g., a human). Certain factors mayinfluence the dosage and timing required to effectively treat a subject(e.g., the severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and the presence of otherdiseases).

In some embodiments, the subject is further administered an additionaltherapeutic agent (e.g., any of the additional therapeutic agentsdescribed herein). The additional therapeutic agent can be administeredto the subject at substantially the same time as the TLR agonist orpharmaceutical composition comprising it is administered and/or at oneor more other time points. In some embodiments, the additionaltherapeutic agent is formulated together with the TLR agonist (e.g.,using any of the examples of formulations described herein).

In some embodiments, the subject is administered a dose of the TLRagonist at least once a month (e.g., at least twice a month, at leastthree times a month, at least four times a month, at least once a week,at least twice a week, three times a week, once a day, or twice a day).The TLR agonist may be administered to a subject chronically. Chronictreatments include any form of repeated administration for an extendedperiod of time, such as repeated administrations for one or more months,between a month and a year, one or more years, more than five years,more than 10 years, more than 15 years, more than 20 years, more than 25years, more than 30 years, more than 35 years, more than 40 years, morethan 45 years, or longer. Alternatively, or in addition, chronictreatments may be administered. Chronic treatments can involve regularadministrations, for example one or more times a day, one or more timesa week, or one or more times a month. For example, chronic treatment caninclude administration (e.g., intravenous administration) about everytwo weeks (e.g., between about every 10 to 18 days).

A suitable dose may be the amount that is the lowest dose effective toproduce a desired therapeutic effect. Such an effective dose willgenerally depend upon the factors described herein. If desired, aneffective daily dose of TLR agonist can be administered as two, three,four, five, or six or more sub-doses administered separately atappropriate intervals throughout the day, optionally, in unit dosageforms.

In some examples, administration of an TLR agonist using any of thecompositions or devices described herein can result in the onset oftreatment (e.g., a reduction in the number, severity, or duration of oneor more symptoms and/or markers of any of the diseases described herein)or drug-target engagement in a subject within a time period of about 10minutes to about 10 hours, about 10 minutes to about 9 hours, about 10minutes to about 8 hours, about 10 minutes to about 7 hours, about 10minutes to about 6 hours, about 10 minutes to about 5 hours, about 10minutes to about 4.5 hours, about 10 minutes to about 4 hours, about 10minutes to about 3.5 hours, about 10 minutes to about 3 hours, about 10minutes to about 2.5 hours, about 10 minutes to about 2 hours, about 10minutes to about 1.5 hours, about 10 minutes to about 1 hour, about 10minutes to about 55 minutes, about 10 minutes to about 50 minutes, about10 minutes to about 45 minutes, about 10 minutes to about 40 minutes,about 10 minutes to about 35 minutes, about 10 minutes to about 30minutes, about 10 minutes to about 25 minutes, about 10 minutes to about20 minutes, about 10 minutes to about 15 minutes, about 15 minutes toabout 10 hours, about 15 minutes to about 9 hours, about 15 minutes toabout 8 hours, about 15 minutes to about 7 hours, about 15 minutes toabout 6 hours, about 15 minutes to about 5 hours, about 15 minutes toabout 4.5 hours, about 15 minutes to about 4 hours, about 15 minutes toabout 3.5 hours, about 15 minutes to about 3 hours, about 15 minutes toabout 2.5 hours, about 15 minutes to about 2 hours, about 15 minutes toabout 1.5 hours, about 15 minutes to about 1 hour, about 15 minutes toabout 55 minutes, about 15 minutes to about 50 minutes, about 15 minutesto about 45 minutes, about 15 minutes to about 40 minutes, about 15minutes to about 35 minutes, about 15 minutes to about 30 minutes, about15 minutes to about 25 minutes, about 15 minutes to about 20 minutes,about 20 minutes to about 10 hours, about 20 minutes to about 9 hours,about 20 minutes to about 8 hours, about 20 minutes to about 7 hours,about 20 minutes to about 6 hours, about 20 minutes to about 5 hours,about 20 minutes to about 4.5 hours, about 20 minutes to about 4 hours,about 20 minutes to about 3.5 hours, about 20 minutes to about 3 hours,about 20 minutes to about 2.5 hours, about 20 minutes to about 2 hours,about 20 minutes to about 1.5 hours, about 20 minutes to about 1 hour,about 20 minutes to about 55 minutes, about 20 minutes to about 50minutes, about 20 minutes to about 45 minutes, about 20 minutes to about40 minutes, about 20 minutes to about 35 minutes, about 20 minutes toabout 30 minutes, about 20 minutes to about 25 minutes, about 25 minutesto about 10 hours, about 25 minutes to about 9 hours, about 25 minutesto about 8 hours, about 25 minutes to about 7 hours, about 25 minutes toabout 6 hours, about 25 minutes to about 5 hours, about 25 minutes toabout 4.5 hours, about 25 minutes to about 4 hours, about 25 minutes toabout 3.5 hours, about 25 minutes to about 3 hours, about 25 minutes toabout 2.5 hours, about 25 minutes to about 2 hours, about 25 minutes toabout 1.5 hours, about 25 minutes to about 1 hour, about 25 minutes toabout 55 minutes, about 25 minutes to about 50 minutes, about 25 minutesto about 45 minutes, about 25 minutes to about 40 minutes, about 25minutes to about 35 minutes, about 25 minutes to about 30 minutes, about30 minutes to about 10 hours, about 30 minutes to about 9 hours, about30 minutes to about 8 hours, about 30 minutes to about 7 hours, about 30minutes to about 6 hours, about 30 minutes to about 5 hours, about 30minutes to about 4.5 hours, about 30 minutes to about 4 hours, about 30minutes to about 3.5 hours, about 30 minutes to about 3 hours, about 30minutes to about 2.5 hours, about 30 minutes to about 2 hours, about 30minutes to about 1.5 hours, about 30 minutes to about 1 hour, about 30minutes to about 55 minutes, about 30 minutes to about 50 minutes, about30 minutes to about 45 minutes, about 30 minutes to about 40 minutes,about 30 minutes to about 35 minutes, about 35 minutes to about 10hours, about 35 minutes to about 9 hours, about 35 minutes to about 8hours, about 35 minutes to about 7 hours, about 35 minutes to about 6hours, about 35 minutes to about 5 hours, about 35 minutes to about 4.5hours, about 35 minutes to about 4 hours, about 35 minutes to about 3.5hours, about 35 minutes to about 3 hours, about 35 minutes to about 2.5hours, about 35 minutes to about 2 hours, about 35 minutes to about 1.5hours, about 35 minutes to about 1 hour, about 35 minutes to about 55minutes, about 35 minutes to about 50 minutes, about 35 minutes to about45 minutes, about 35 minutes to about 40 minutes, about 40 minutes toabout 10 hours, about 40 minutes to about 9 hours, about 40 minutes toabout 8 hours, about 40 minutes to about 7 hours, about 40 minutes toabout 6 hours, about 40 minutes to about 5 hours, about 40 minutes toabout 4.5 hours, about 40 minutes to about 4 hours, about 40 minutes toabout 3.5 hours, about 40 minutes to about 3 hours, about 40 minutes toabout 2.5 hours, about 40 minutes to about 2 hours, about 40 minutes toabout 1.5 hours, about 40 minutes to about 1 hour, about 40 minutes toabout 55 minutes, about 40 minutes to about 50 minutes, about 40 minutesto about 45 minutes, about 45 minutes to about 10 hours, about 45minutes to about 9 hours, about 45 minutes to about 8 hours, about 45minutes to about 7 hours, about 45 minutes to about 6 hours, about 45minutes to about 5 hours, about 45 minutes to about 4.5 hours, about 45minutes to about 4 hours, about 45 minutes to about 3.5 hours, about 45minutes to about 3 hours, about 45 minutes to about 2.5 hours, about 45minutes to about 2 hours, about 45 minutes to about 1.5 hours, about 45minutes to about 1 hour, about 45 minutes to about 55 minutes, about 45minutes to about 50 minutes, about 50 minutes to about 10 hours, about50 minutes to about 9 hours, about 50 minutes to about 8 hours, about 50minutes to about 7 hours, about 50 minutes to about 6 hours, about 50minutes to about 5 hours, about 50 minutes to about 4.5 hours, about 50minutes to about 4 hours, about 50 minutes to about 3.5 hours, about 50minutes to about 3 hours, about 50 minutes to about 2.5 hours, about 50minutes to about 2 hours, about 50 minutes to about 1.5 hours, about 50minutes to about 1 hour, about 50 minutes to about 55 minutes, about 55minutes to about 10 hours, about 55 minutes to about 9 hours, about 55minutes to about 8 hours, about 55 minutes to about 7 hours, about 55minutes to about 6 hours, about 55 minutes to about 5 hours, about 55minutes to about 4.5 hours, about 55 minutes to about 4 hours, about 55minutes to about 3.5 hours, about 55 minutes to about 3 hours, about 55minutes to about 2.5 hours, about 55 minutes to about 2 hours, about 55minutes to about 1.5 hours, about 55 minutes to about 1 hour, about 1hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour toabout 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6hours, about 1 hour to about 5 hours, about 1 hour to about 4.5 hours,about 1 hour to about 4 hours, about 1 hour to about 3.5 hours, about 1hour to about 3 hours, about 1 hour to about 2.5 hours, about 1 hour toabout 2 hours, about 1 hour to about 1.5 hours, about 1.5 hours to about10 hours, about 1.5 hours to about 9 hours, about 1.5 hours to about 8hours, about 1.5 hours to about 7 hours, about 1.5 hours to about 6hours, about 1.5 hours to about 5 hours, about 1.5 hours to about 4.5hours, about 1.5 hours to about 4 hours, about 1.5 hours to about 3.5hours, about 1.5 hours to about 3 hours, about 1.5 hours to about 2.5hours, about 1.5 hours to about 2 hours, about 2 hours to about 10hours, about 2 hours to about 9 hours, about 2 hours to about 8 hours,about 2 hours to about 7 hours, about 2 hours to about 6 hours, about 2hours to about 5 hours, about 2 hours to about 4.5 hours, about 2 hoursto about 4 hours, about 2 hours to about 3.5 hours, about 2 hours toabout 3 hours, about 2 hours to about 2.5 hours, about 2.5 hours toabout 10 hours, about 2.5 hours to about 9 hours, about 2.5 hours toabout 8 hours, about 2.5 hours to about 7 hours, about 2.5 hours toabout 6 hours, about 2.5 hours to about 5 hours, about 2.5 hours toabout 4.5 hours, about 2.5 hours to about 4 hours, about 2.5 hours toabout 3.5 hours, about 2.5 hours to about 3 hours, about 3 hours toabout 10 hours, about 3 hours to about 9 hours, about 3 hours to about 8hours, about 3 hours to about 7 hours, about 3 hours to about 6 hours,about 3 hours to about 5 hours, about 3 hours to about 4.5 hours, about3 hours to about 4 hours, about 3 hours to about 3.5 hours, about 3.5hours to about 10 hours, about 3.5 hours to about 9 hours, about 3.5hours to about 8 hours, about 3.5 hours to about 7 hours, about 3.5hours to about 6 hours, about 3.5 hours to about 5 hours, about 3.5hours to about 4.5 hours, about 3.5 hours to about 4 hours, about 4hours to about 10 hours, about 4 hours to about 9 hours, about 4 hoursto about 8 hours, about 4 hours to about 7 hours, about 4 hours to about6 hours, about 4 hours to about 5 hours, about 4 hours to about 4.5hours, about 4.5 hours to about 10 hours, about 4.5 hours to about 9hours, about 4.5 hours to about 8 hours, about 4.5 hours to about 7hours, about 4.5 hours to about 6 hours, about 4.5 hours to about 5hours, about 5 hours to about 10 hours, about 5 hours to about 9 hours,about 5 hours to about 8 hours, about 5 hours to about 7 hours, about 5hours to about 6 hours, about 6 hours to about 10 hours, about 6 hoursto about 9 hours, about 6 hours to about 8 hours, about 6 hours to about7 hours, about 7 hours to about 10 hours, about 7 hours to about 9hours, about 7 hours to about 8 hours, about 8 hours to about 10 hours,about 8 hours to about 9 hours, or about 9 hours to about 10 hours ofadministration of a dose of an TLR agonist using any of the devices orcompositions described herein. Drug-target engagement may be determined,for example, as disclosed in Simon G M, Niphakis M J, Cravat B F, Naturechemical biology. 2013; 9(4):200-205, incorporated by reference hereinin its entirety.

In some embodiments, administration of an TLR agonist using any of thedevices or compositions described herein can provide for treatment(e.g., a reduction in the number, severity, and/or duration of one ormore symptoms and/or markers of any of the disorders described herein ina subject) for a time period of between about 1 hour to about 30 days,about 1 hour to about 28 days, about 1 hour to about 26 days, about 1hour to about 24 days, about 1 hour to about 22 days, about 1 hour toabout 20 days, about 1 hour to about 18 days, about 1 hour to about 16days, about 1 hour to about 14 days, about 1 hour to about 12 days,about 1 hour to about 10 days, about 1 hour to about 8 days, about 1hour to about 6 days, about 1 hour to about 5 days, about 1 hour toabout 4 days, about 1 hour to about 3 days, about 1 hour to about 2days, about 1 hour to about 1 day, about 1 hour to about 12 hours, about1 hour to about 6 hours, about 1 hour to about 3 hours, about 3 hours toabout 30 days, about 3 hours to about 28 days, about 3 hours to about 26days, about 3 hours to about 24 days, about 3 hours to about 22 days,about 3 hours to about 20 days, about 3 hours to about 18 days, about 3hours to about 16 days, about 3 hours to about 14 days, about 3 hours toabout 12 days, about 3 hours to about 10 days, about 3 hours to about 8days, about 3 hours to about 6 days, about 3 hours to about 5 days,about 3 hours to about 4 days, about 3 hours to about 3 days, about 3hours to about 2 days, about 3 hours to about 1 day, about 3 hours toabout 12 hours, about 3 hours to about 6 hours, about 6 hours to about30 days, about 6 hours to about 28 days, about 6 hours to about 26 days,about 6 hours to about 24 days, about 6 hours to about 22 days, about 6hours to about 20 days, about 6 hours to about 18 days, about 6 hours toabout 16 days, about 6 hours to about 14 days, about 6 hours to about 12days, about 6 hours to about 10 days, about 6 hours to about 8 days,about 6 hours to about 6 days, about 6 hours to about 5 days, about 6hours to about 4 days, about 6 hours to about 3 days, about 6 hours toabout 2 days, about 6 hours to about 1 day, about 6 hours to about 12hours, about 12 hours to about 30 days, about 12 hours to about 28 days,about 12 hours to about 26 days, about 12 hours to about 24 days, about12 hours to about 22 days, about 12 hours to about 20 days, about 12hours to about 18 days, about 12 hours to about 16 days, about 12 hoursto about 14 days, about 12 hours to about 12 days, about 12 hours toabout 10 days, about 12 hours to about 8 days, about 12 hours to about 6days, about 12 hours to about 5 days, about 12 hours to about 4 days,about 12 hours to about 3 days, about 12 hours to about 2 days, about 12hours to about 1 day, about 1 day to about 30 days, about 1 day to about28 days, about 1 day to about 26 days, about 1 day to about 24 days,about 1 day to about 22 days, about 1 day to about 20 days, about 1 dayto about 18 days, about 1 day to about 16 days, about 1 day to about 14days, about 1 day to about 12 days, about 1 day to about 10 days, about1 day to about 8 days, about 1 day to about 6 days, about 1 day to about5 days, about 1 day to about 4 days, about 1 day to about 3 days, about1 day to about 2 days, about 2 days to about 30 days, about 2 days toabout 28 days, about 2 days to about 26 days, about 2 days to about 24days, about 2 days to about 22 days, about 2 days to about 20 days,about 2 days to about 18 days, about 2 days to about 16 days, about 2days to about 14 days, about 2 days to about 12 days, about 2 days toabout 10 days, about 2 days to about 8 days, about 2 days to about 6days, about 2 days to about 5 days, about 2 days to about 4 days, about2 days to about 3 days, about 3 days to about 30 days, about 3 days toabout 28 days, about 3 days to about 26 days, about 3 days to about 24days, about 3 days to about 22 days, about 3 days to about 20 days,about 3 days to about 18 days, about 3 days to about 16 days, about 3days to about 14 days, about 3 days to about 12 days, about 3 days toabout 10 days, about 3 days to about 8 days, about 3 days to about 6days, about 3 days to about 5 days, about 3 days to about 4 days, about4 days to about 30 days, about 4 days to about 28 days, about 4 days toabout 26 days, about 4 days to about 24 days, about 4 days to about 22days, about 4 days to about 20 days, about 4 days to about 18 days,about 4 days to about 16 days, about 4 days to about 14 days, about 4days to about 12 days, about 4 days to about 10 days, about 4 days toabout 8 days, about 4 days to about 6 days, about 4 days to about 5days, about 5 days to about 30 days, about 5 days to about 28 days,about 5 days to about 26 days, about 5 days to about 24 days, about 5days to about 22 days, about 5 days to about 20 days, about 5 days toabout 18 days, about 5 days to about 16 days, about 5 days to about 14days, about 5 days to about 12 days, about 5 days to about 10 days,about 5 days to about 8 days, about 5 days to about 6 days, about 6 daysto about 30 days, about 6 days to about 28 days, about 6 days to about26 days, about 6 days to about 24 days, about 6 days to about 22 days,about 6 days to about 20 days, about 6 days to about 18 days, about 6days to about 16 days, about 6 days to about 14 days, about 6 days toabout 12 days, about 6 days to about 10 days, about 6 days to about 8days, about 8 days to about 30 days, about 8 days to about 28 days,about 8 days to about 26 days, about 8 days to about 24 days, about 8days to about 22 days, about 8 days to about 20 days, about 8 days toabout 18 days, about 8 days to about 16 days, about 8 days to about 14days, about 8 days to about 12 days, about 8 days to about 10 days,about 10 days to about 30 days, about 10 days to about 28 days, about 10days to about 26 days, about 10 days to about 24 days, about 10 days toabout 22 days, about 10 days to about 20 days, about 10 days to about 18days, about 10 days to about 16 days, about 10 days to about 14 days,about 10 days to about 12 days, about 12 days to about 30 days, about 12days to about 28 days, about 12 days to about 26 days, about 12 days toabout 24 days, about 12 days to about 22 days, about 12 days to about 20days, about 12 days to about 18 days, about 12 days to about 16 days,about 12 days to about 14 days, about 14 days to about 30 days, about 14days to about 28 days, about 14 days to about 26 days, about 14 days toabout 24 days, about 14 days to about 22 days, about 14 days to about 20days, about 14 days to about 18 days, about 14 days to about 16 days,about 16 days to about 30 days, about 16 days to about 28 days, about 16days to about 26 days, about 16 days to about 24 days, about 16 days toabout 22 days, about 16 days to about 20 days, about 16 days to about 18days, about 18 days to about 30 days, about 18 days to about 28 days,about 18 days to about 26 days, about 18 days to about 24 days, about 18days to about 22 days, about 18 days to about 20 days, about 20 days toabout 30 days, about 20 days to about 28 days, about 20 days to about 26days, about 20 days to about 24 days, about 20 days to about 22 days,about 22 days to about 30 days, about 22 days to about 28 days, about 22days to about 26 days, about 22 days to about 24 days, about 24 days toabout 30 days, about 24 days to about 28 days, about 24 days to about 26days, about 26 days to about 30 days, about 26 days to about 28 days, orabout 28 days to about 30 days in a subject following firstadministration of an TLR agonist using any of the compositions ordevices described herein. Non-limiting examples of symptoms and/ormarkers of a disease described herein are described below.

For example, treatment can result in a decrease (e.g., about 1% to about99% decrease, about 1% to about 95% decrease, about 1% to about 90%decrease, about 1% to about 85% decrease, about 1% to about 80%decrease, about 1% to about 75% decrease, about 1% to about 70%decrease, about 1% to about 65% decrease, about 1% to about 60%decrease, about 1% to about 55% decrease, about 1% to about 50%decrease, about 1% to about 45% decrease, about 1% to about 40%decrease, about 1% to about 35% decrease, about 1% to about 30%decrease, about 1% to about 25% decrease, about 1% to about 20%decrease, about 1% to about 15% decrease, about 1% to about 10%decrease, about 1% to about 5% decrease, about 5% to about 99% decrease,about 5% to about 95% decrease, about 5% to about 90% decrease, about 5%to about 85% decrease, about 5% to about 80% decrease, about 5% to about75% decrease, about 5% to about 70% decrease, about 5% to about 65%decrease, about 5% to about 60% decrease, about 5% to about 55%decrease, about 5% to about 50% decrease, about 5% to about 45%decrease, about 5% to about 40% decrease, about 5% to about 35%decrease, about 5% to about 30% decrease, about 5% to about 25%decrease, about 5% to about 20% decrease, about 5% to about 15%decrease, about 5% to about 10% decrease, about 10% to about 99%decrease, about 10% to about 95% decrease, about 10% to about 90%decrease, about 10% to about 85% decrease, about 10% to about 80%decrease, about 10% to about 75% decrease, about 10% to about 70%decrease, about 10% to about 65% decrease, about 10% to about 60%decrease, about 10% to about 55% decrease, about 10% to about 50%decrease, about 10% to about 45% decrease, about 10% to about 40%decrease, about 10% to about 35% decrease, about 10% to about 30%decrease, about 10% to about 25% decrease, about 10% to about 20%decrease, about 10% to about 15% decrease, about 15% to about 99%decrease, about 15% to about 95% decrease, about 15% to about 90%decrease, about 15% to about 85% decrease, about 15% to about 80%decrease, about 15% to about 75% decrease, about 15% to about 70%decrease, about 15% to about 65% decrease, about 15% to about 60%decrease, about 15% to about 55% decrease, about 15% to about 50%decrease, about 15% to about 45% decrease, about 15% to about 40%decrease, about 15% to about 35% decrease, about 15% to about 30%decrease, about 15% to about 25% decrease, about 15% to about 20%decrease, about 20% to about 99% decrease, about 20% to about 95%decrease, about 20% to about 90% decrease, about 20% to about 85%decrease, about 20% to about 80% decrease, about 20% to about 75%decrease, about 20% to about 70% decrease, about 20% to about 65%decrease, about 20% to about 60% decrease, about 20% to about 55%decrease, about 20% to about 50% decrease, about 20% to about 45%decrease, about 20% to about 40% decrease, about 20% to about 35%decrease, about 20% to about 30% decrease, about 20% to about 25%decrease, about 25% to about 99% decrease, about 25% to about 95%decrease, about 25% to about 90% decrease, about 25% to about 85%decrease, about 25% to about 80% decrease, about 25% to about 75%decrease, about 25% to about 70% decrease, about 25% to about 65%decrease, about 25% to about 60% decrease, about 25% to about 55%decrease, about 25% to about 50% decrease, about 25% to about 45%decrease, about 25% to about 40% decrease, about 25% to about 35%decrease, about 25% to about 30% decrease, about 30% to about 99%decrease, about 30% to about 95% decrease, about 30% to about 90%decrease, about 30% to about 85% decrease, about 30% to about 80%decrease, about 30% to about 75% decrease, about 30% to about 70%decrease, about 30% to about 65% decrease, about 30% to about 60%decrease, about 30% to about 55% decrease, about 30% to about 50%decrease, about 30% to about 45% decrease, about 30% to about 40%decrease, about 30% to about 35% decrease, about 35% to about 99%decrease, about 35% to about 95% decrease, about 35% to about 90%decrease, about 35% to about 85% decrease, about 35% to about 80%decrease, about 35% to about 75% decrease, about 35% to about 70%decrease, about 35% to about 65% decrease, about 35% to about 60%decrease, about 35% to about 55% decrease, about 35% to about 50%decrease, about 35% to about 45% decrease, about 35% to about 40%decrease, about 40% to about 99% decrease, about 40% to about 95%decrease, about 40% to about 90% decrease, about 40% to about 85%decrease, about 40% to about 80% decrease, about 40% to about 75%decrease, about 40% to about 70% decrease, about 40% to about 65%decrease, about 40% to about 60% decrease, about 40% to about 55%decrease, about 40% to about 50% decrease, about 40% to about 45%decrease, about 45% to about 99% decrease, about 45% to about 95%decrease, about 45% to about 90% decrease, about 45% to about 85%decrease, about 45% to about 80% decrease, about 45% to about 75%decrease, about 45% to about 70% decrease, about 45% to about 65%decrease, about 45% to about 60% decrease, about 45% to about 55%decrease, about 45% to about 50% decrease, about 50% to about 99%decrease, about 50% to about 95% decrease, about 50% to about 90%decrease, about 50% to about 85% decrease, about 50% to about 80%decrease, about 50% to about 75% decrease, about 50% to about 70%decrease, about 50% to about 65% decrease, about 50% to about 60%decrease, about 50% to about 55% decrease, about 55% to about 99%decrease, about 55% to about 95% decrease, about 55% to about 90%decrease, about 55% to about 85% decrease, about 55% to about 80%decrease, about 55% to about 75% decrease, about 55% to about 70%decrease, about 55% to about 65% decrease, about 55% to about 60%decrease, about 60% to about 99% decrease, about 60% to about 95%decrease, about 60% to about 90% decrease, about 60% to about 85%decrease, about 60% to about 80% decrease, about 60% to about 75%decrease, about 60% to about 70% decrease, about 60% to about 65%decrease, about 65% to about 99% decrease, about 65% to about 95%decrease, about 65% to about 90% decrease, about 65% to about 85%decrease, about 65% to about 80% decrease, about 65% to about 75%decrease, about 65% to about 70% decrease, about 70% to about 99%decrease, about 70% to about 95% decrease, about 70% to about 90%decrease, about 70% to about 85% decrease, about 70% to about 80%decrease, about 70% to about 75% decrease, about 75% to about 99%decrease, about 75% to about 95% decrease, about 75% to about 90%decrease, about 75% to about 85% decrease, about 75% to about 80%decrease, about 80% to about 99% decrease, about 80% to about 95%decrease, about 80% to about 90% decrease, about 80% to about 85%decrease, about 85% to about 99% decrease, about 85% to about 95%decrease, about 85% to about 90% decrease, about 90% to about 99%decrease, about 90% to about 95% decrease, or about 95% to about 99%decrease) in one or more (e.g., two, three, four, five, six, seven,eight, or nine) of: the level of interferon-γ in GI tissue, the level ofIL-1β in GI tissue, the level of IL-6 in GI tissue, the level of IL-22in GI tissue, the level of IL-17A in the GI tissue, the level of TNFα inGI tissue, the level of IL-2 in GI tissue, and endoscopy score in asubject (e.g., as compared to the level in the subject prior totreatment or compared to a subject or population of subjects having asimilar disease but receiving a placebo or a different treatment) (e.g.,for a time period of between about 1 hour to about 30 days (e.g., or anyof the subranges herein) following the first administration of an TLRagonist using any of the compositions or devices described herein. Asused herein, “GI tissue” refers to tissue in the gastrointestinal (GI)tract, such as tissue in one or more of duodenum, jejunum, ileum, cecum,ascending colon, transverse colon, descending colon, sigmoid colon, andrectum, more particularly in the proximal portion of one or more ofduodenum, jejunum, ileum, cecum, ascending colon, transverse colon,descending colon, and sigmoid colon, or in the distal portion of one ormore of duodenum, jejunum, ileum, cecum, ascending colon, transversecolon, descending colon, and sigmoid colon. The GI tissue may be, forexample, GI tissue proximate to one or more sites of disease. Exemplarymethods for determining the endoscopy score are described herein andother methods for determining the endoscopy score are known in the art.Exemplary methods for determining the levels of interferon-γ, IL-1β,IL-6, IL-22, IL-17A, TNFα, and IL-2 are described herein. Additionalmethods for determining the levels of these cytokines are known in theart.

In some examples, treatment can result in an increase (e.g., about 1% toabout 500% increase, about 1% to about 400% increase, about 1% to about300% increase, about 1% to about 200% increase, about 1% to about 150%increase, about 1% to about 100% increase, about 1% to about 90%increase, about 1% to about 80% increase, about 1% to about 70%increase, about 1% to about 60% increase, about 1% to about 50%increase, about 1% to about 40% increase, about 1% to about 30%increase, about 1% to about 20% increase, about 1% to about 10%increase, a 10% to about 500% increase, about 10% to about 400%increase, about 10% to about 300% increase, about 10% to about 200%increase, about 10% to about 150% increase, about 10% to about 100%increase, about 10% to about 90% increase, about 10% to about 80%increase, about 10% to about 70% increase, about 10% to about 60%increase, about 10% to about 50% increase, about 10% to about 40%increase, about 10% to about 30% increase, about 10% to about 20%increase, about 20% to about 500% increase, about 20% to about 400%increase, about 20% to about 300% increase, about 20% to about 200%increase, about 20% to about 150% increase, about 20% to about 100%increase, about 20% to about 90% increase, about 20% to about 80%increase, about 20% to about 70% increase, about 20% to about 60%increase, about 20% to about 50% increase, about 20% to about 40%increase, about 20% to about 30% increase, about 30% to about 500%increase, about 30% to about 400% increase, about 30% to about 300%increase, about 30% to about 200% increase, about 30% to about 150%increase, about 30% to about 100% increase, about 30% to about 90%increase, about 30% to about 80% increase, about 30% to about 70%increase, about 30% to about 60% increase, about 30% to about 50%increase, about 30% to about 40% increase, about 40% to about 500%increase, about 40% to about 400% increase, about 40% to about 300%increase, about 40% to about 200% increase, about 40% to about 150%increase, about 40% to about 100% increase, about 40% to about 90%increase, about 40% to about 80% increase, about 40% to about 70%increase, about 40% to about 60% increase, about 40% to about 50%increase, about 50% to about 500% increase, about 50% to about 400%increase, about 50% to about 300% increase, about 50% to about 200%increase, about 50% to about 150% increase, about 50% to about 100%increase, about 50% to about 90% increase, about 50% to about 80%increase, about 50% to about 70% increase, about 50% to about 60%increase, about 60% to about 500% increase, about 60% to about 400%increase, about 60% to about 300% increase, about 60% to about 200%increase, about 60% to about 150% increase, about 60% to about 100%increase, about 60% to about 90% increase, about 60% to about 80%increase, about 60% to about 70% increase, about 70% to about 500%increase, about 70% to about 400% increase, about 70% to about 300%increase, about 70% to about 200% increase, about 70% to about 150%increase, about 70% to about 100% increase, about 70% to about 90%increase, about 70% to about 80% increase, about 80% to about 500%increase, about 80% to about 400% increase, about 80% to about 300%increase, about 80% to about 200% increase, about 80% to about 150%increase, about 80% to about 100% increase, about 80% to about 90%increase, about 90% to about 500% increase, about 90% to about 400%increase, about 90% to about 300% increase, about 90% to about 200%increase, about 90% to about 150% increase, about 90% to about 100%increase, about 100% to about 500% increase, about 100% to about 400%increase, about 100% to about 300% increase, about 100% to about 200%increase, about 100% to about 150% increase, about 150% to about 500%increase, about 150% to about 400% increase, about 150% to about 300%increase, about 150% to about 200% increase, about 200% to about 500%increase, about 200% to about 400% increase, about 200% to about 300%increase, about 300% to about 500% increase, about 300% to about 400%increase, or about 400% to about 500% increase) in one or both of stoolconsistency score and weight of a subject (e.g., as compared to thelevel in the subject prior to treatment or compared to a subject orpopulation of subjects having a similar disease but receiving a placeboor a different treatment) (e.g., for a time period of between about 1hour to about 30 days (e.g., or any of the subranges herein) followingthe first administration of an TLR agonist using any of the compositionsor devices described herein. Exemplary methods for determining stoolconsistency score are described herein. Additional methods fordetermining a stool consistency score are known in the art.

Accordingly, in some embodiments, a method of treatment disclosed hereinincludes determining the level of a marker at the location of disease ina subject (e.g., either before and/or after administration of thedevice). In some embodiments, the marker is a biomarker and the methodof treatment disclosed herein comprises determining that the level of abiomarker at the location of disease is a subject followingadministration of the device is decreased as compared to the level ofthe biomarker at the same location of disease in a subject either beforeadministration or at the same time point following systemicadministration of an equal amount of the TLR agonist. In some examples,the level of the biomarker at the same location of disease followingadministration of the device is 1% decreased to 99% decreased ascompared to the level of the biomarker at the same location of diseasein a subject either before administration or at the same time pointfollowing systemic administration of an equal amount of the TLR agonist.In some embodiments, the level of the marker is one or more of: thelevel of interferon-γ in GI tissue, the level of IL-17A in the GItissue, the level of TNFα in the GI tissue, the level of IL-2 in the GItissue, and the endoscopy score in a subject.

In some embodiments, the method of treatment disclosed herein includesdetermining that the level of a marker at a time point followingadministration of a device is lower than the level of the marker at atime point following administration of the device is lower than thelevel of the marker in a subject prior to administration of the deviceor in a subject at substantially the same time point following systemicadministration of an equal amount of the TLR agonist. In some examples,the level of the marker following administration of the device is 1%decreased to 99% decreased as compared to the level of the marker in asubject prior to administration of the device or in a subject at thesame time point following systemic administration of an equal amount ofthe TLR agonist. In some examples, a method of treatment disclosedherein includes determining the level of the biomarker at the locationof disease in a subject within a time period of about 10 minutes to 10hours following administration of the device.

In some embodiments, a method of treatment described herein includes:(i) determining the ratio R_(B) of the level L_(1B) of a biomarker atthe location of disease at a first time point following administrationof the device and the level L_(2B) of the biomarker at the same locationof disease in a subject at substantially the same time point followingsystemic administration of an equal amount of the TLR agonist; (ii)determining the ratio of R_(D) of the level of L_(1D) of the TLR agonistat the same location and the substantially the same time point as in (i)and the level L_(2D) of the TLR agonist at the same location of diseasein a subject at substantially the same time point following systemicadministration of an equal amount of the TLR agonist; and (iii)determining the ratio of R_(B)/R_(D).

In some embodiments, a method of treatment disclosed herein can include:(i) determining the ratio R_(B) of the level L_(1B) of a biomarker atthe location of disease at a time point following administration of thedevice and the level L_(2B) of the biomarker at the same location ofdisease in a subject at substantially the same time point followingsystemic administration of an equal amount of the TLR agonist; (ii)determining the ratio R_(D) of the level L_(1D) of the TLR agonist atthe same location and at substantially the time point as in (i) and thelevel L2D of the TLR agonist in a subject at the same location ofdisease at substantially the same time point following systemicadministration of an equal amount of the TLR agonist; and (iii)determining the product R_(B)×R_(D).

In some embodiments, a method of treatment disclosed herein can includedetermining that the level of a marker in a subject at a time pointfollowing administration of the device is elevated as compared to alevel of the marker in a subject prior to administration of the deviceor a level at substantially the same time point in a subject followingsystemic administration of an equal amount of the TLR agonist. In someexamples, the level of the marker at a time point followingadministration of the device is 1% increased or 400% increased ascompared to the level of the marker in a subject prior to administrationof the device or a level at substantially the same time point in asubject following systemic administration of an equal amount of the TLRagonist. In some examples, the level of the marker is one or more ofsubject weight and stool consistency (e.g., stool consistency score). Insome examples, a method of treatment disclosed herein includesdetermining the level of the marker in a subject within a period ofabout 10 minutes to about 10 hours following administration of thedevice.

In some embodiments, a method of treatment disclosed herein can includedetermining the level of a marker in a subject's blood, serum or plasma.

An illustrative list of examples of biomarkers for GI disorders includesinterferon-γ, IL-1β, IL-6, IL-22, IL-17A, TNFα, IL-2, memory cells(CD44⁺CD45RB⁻CD4⁺ cells); α4β7; VEGF; ICAM; VCAM; SAA; Calprotectin;lactoferrin; FGF2; TGFb; ANG-1; ANG-2; PLGF; Biologics (Infliximab;Humira; Stelara; Vedolizumab; Simponi; Jak inhibitors; Others); EGF;IL12/23p40; GMCSF; A4 B7; AeB7; CRP; SAA; ICAM; VCAM; AREG; EREG;HB-EGF; HRG; BTC; TGFα; SCF; TWEAK; MMP-9; MMP-6; Ceacam CD66; IL10;ADA; Madcam-1; CD166 (AL CAM); FGF2; FGF7; FGF9; FGF19; ANCAAntineutrophil cytoplasmic antibody; ASCAA Anti-Saccharomyces CerevisiaeAntibody IgA; ASCAG Anti-Saccharomyces Cerevisiae Antibody IgG; CBir1Anti-Clostridium cluster XIVa flagellin CBir1 antibody; A4-Fla2Anti-Clostridium cluster XIVa flagellin 2 antibody; FlaXAnti-Clostridium cluster XIVa flagellin X antibody; OmpCAnti-Escherichia coli Outer Membrane Protein C; ANCA PerinuclearAntiNeutrophil Cytoplasmic Antibody; AREG Amphiregulin Protein; BTCBetacellulin Protein; EGF Epidermal Growth Factor EREG

Epiregulin Protein; HBEGF Heparin Binding Epidermal Growth Factors; HGFHepatocyte Growth Factor; HRG Neuregulin-1; TGFA Transforming GrowthFactor alpha; CRP C-Reactive Protein; SAA Serum Amyloid A; ICAM-1Intercellular Adhesion Molecule 1; VCAM-1 Vascular Cell AdhesionMolecule 1; fibroblasts underlying the intestinal epithelium; and HGF.

In some embodiments, a marker is an IBD biomarker, such as, for example:anti-glycan; anti-Saccharomyces cerevisiae (ASCA); anti-laminaribioside(ALCA); anti-chitobioside (ACCA); anti-mannobioside (AMCA);anti-laminarin (anti-L); anti-chitin (anti-C) antibodies: anti-outermembrane porin C (anti-OmpC), anti-Cbir1 flagellin; anti-12 antibody;autoantibodies targeting the exocrine pancreas (PAB); and perinuclearanti-neutrophil antibody (pANCA); and calprotectin.

In some embodiments, a biomarker is associated with membrane repair,fibrosis, angiogenesis. In certain embodiments, a biomarker is aninflammatory biomarker, an anti-inflammatory biomarker, an MMPbiomarker, an immune marker, or a TNF pathway biomarker. In someembodiments, a biomarker is gut specific.

For tissue samples, HER2 can be used as a biomarker relating tocytotoxic T cells. Additionally, other cytokine levels can be used asbiomarkers in tissue (e.g., phospho STAT 1, STAT 3 and STAT 5), inplasma (e.g., VEGF, VCAM, ICAM, IL-6), or both.

In some embodiments, the biomarkers include one or more immunoglobulins,such as, for example, immunoglobulin M (IgM), immunoglobulin D (IgD),immunoglobulin G (IgG), immunoglobulin E (IgE) and/or immunoglobulin A(IgA). In some embodiments, IgM is a biomarker of infection and/orinflammation. In some embodiments, IgD is a biomarker of autoimmunedisease. In some embodiments, IgG is a biomarker of Alzheimer's diseaseand/or for cancer. In some embodiments, IgE is a biomarker of asthmaand/or allergen immunotherapy. In some embodiments, IgA is a biomarkerof kidney disease.

In some embodiments, the biomarker is High Sensitivity C-reactiveProtein (hsCRP); 7 α-hydroxy-4-cholesten-3-one (7C4); Anti-EndomysialIgA (EMA IgA); Anti-Human Tissue Transglutaminase IgA (tTG IgA); TotalSerum IgA by Nephelometry; Fecal

Calprotectin; or Fecal Gastrointestinal Pathogens.

In some embodiments, the biomarker is

a) an anti-gliadin IgA antibody, an anti-gliadin IgG antibody, ananti-tissue transglutaminase (tTG) antibody, an anti-endomysialantibody;

b)i) a serological marker that is ASCA-A, ASCA-G, ANCA, pANCA, anti-OmpCantibody, anti-CBir1 antibody, anti-FlaX antibody, or anti-A4-Fla2antibody;

b)ii) an inflammation marker that is VEGF, ICAM, VCAM, SAA, or CRP;

b)iii) the genotype of the genetic markers ATG16L1, ECM1, NKX2-3, orSTAT3;

c) a bacterial antigen antibody marker;

d) a mast cell marker;

e) an inflammatory cell marker;

f) a bile acid malabsorption (BAM) marker;

g) a kynurenine marker;

or

h) a serotonin marker.

In some embodiments, the bacterial antigen antibody marker is selectedfrom the group consisting of an anti-Fla1 antibody, anti-Fla2 antibody,anti-FlaA antibody, anti-FliC antibody, anti-FliC2 antibody, anti-FliC3antibody, anti-YBaN1 antibody, anti-ECFliC antibody, anti-Ec0FliCantibody, anti-SeFljB antibody, anti-CjFlaA antibody, anti-CjFlaBantibody, anti-SfFliC antibody, anti-CjCgtA antibody, anti-Cjdmhantibody, anti-CjGT-A antibody, anti-EcYidX antibody, anti-EcEraantibody, anti-EcFrvX antibody, anti-EcGabT antibody, anti-EcYedKantibody, anti-EcYbaN antibody, anti-EcYhgN antibody, anti-RtMagaantibody, anti-RbCpaF antibody, anti-RgPilD antibody, anti-LaFrcantibody, anti-LaEno antibody, anti-LjEFTu antibody, anti-BfOmpaantibody, anti-PrOmpA antibody, anti-Cp10bA antibody, anti-CpSpAantibody, anti-EfSant antibody, anti-LmOsp antibody, anti-SfET-2antibody, anti-Cpatox antibody, anti-Cpbtox antibody, anti-EcSta2antibody, anti-Ec0Stx2A antibody, anti-CjcdtB/C antibody, anti-CdtcdA/Bantibody, and combinations thereof.

In some embodiments, the mast cell marker is selected from the groupconsisting of beta-tryptase, histamine, prostaglandin E2 (PGE2), andcombinations thereof.

In some embodiments, the inflammatory marker is selected from the groupconsisting of CRP, ICAM, VCAM, SAA, GRO.alpha., and combinationsthereof.

In some embodiments, the bile acid malabsorption marker is selected fromthe group consisting of 7α-hydroxy-4-cholesten-3-one, FGF19, and acombination thereof.

In some embodiments, the kynurenine marker is selected from the groupconsisting of kynurenine (K), kynurenic acid (KyA), anthranilic acid(AA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA),xanthurenic acid (XA), quinolinic acid (QA), tryptophan,5-hydroxytryptophan (5-HTP), and combinations thereof.

In some embodiments, the serotonin marker is selected from the groupconsisting of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA),serotonin-O-sulfate, serotonin-O-phosphate, and combinations thereof.

In some embodiments, the biomarker is a biomarker as disclosed in U.S.Pat. No. 9,739,786, incorporated by reference herein in its entirety.

The following markers can be expressed by mesenchymal stem cells (MSC):CD105, CD73, CD90, CD13, CD29, CD44, CD10, Stro-1, CD271, SSEA-4, CD146,CD49f, CD349, GD2, 3G5, SSEA-3, SISD2, Stro-4, MSCA-1, CD56, CD200,PODX1, Sox11, or TM4SF1 (e.g., 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more of suchmarkers), and lack expression of one or more of CD45, CD34, CD14, CD19,and HLA-DR (e.g., lack expression of two or more, three or more, four ormore, or five or more such markers). In some embodiments, MSC canexpress CD105, CD73, and CD90. In some embodiments, MSC can expressCD105, CD73, CD90, CD13, CD29, CD44, and CD10. In some embodiments, MSCcan express CD105, CD73, and CD90 and one or more stemness markers suchas Stro-1, CD271, SSEA-4, CD146, CD49f, CD349, GD2, 3G5, SSEA-3. SISD2,Stro-4, MSCA-1, CD56, CD200, PODX1, Sox11, or TM4SF1. In someembodiments, MSC can express CD105, CD73, CD90, CD13, CD29, CD44, andCD10 and one or more stemness markers such as Stro-1, CD271, SSEA-4,CD146, CD49f, CD349, GD2, 3G5, SSEA-3. SISD2, Stro-4, MSCA-1, CD56,CD200, PODX1, Sox11, or TM4SF1. See, e.g., Lv, et al., Stem Cells, 2014,32:1408-1419.

Intestinal stem cells (ISC) can be positive for one or more markers suchas Musashi-1 (Msi-1), Asc12, Bmi-1, Doublecortin andCa2+/calmodulin-dependent kinase-like 1 (DCAMKL1), and Leucin-richrepeat-containing G-protein-coupled receptor 5 (Lgrs). See, e.g.,Mohamed, et al., Cytotechnology, 2015 67(2): 177-189.

Any of the foregoing biomarkers can be used as a biomarker for one ormore of other conditions as appropriate.

In some embodiments of the methods herein, the methods comprisedetermining the time period of onset of treatment followingadministration of the device.

Combination Therapy

The TLR agonists disclosed herein may be optionally be used withadditional agents in the treatment of the diseases disclosed herein.Nonlimiting examples of such agents for treating or preventinginflammatory bowel disease in such adjunct therapy (e.g., Crohn'sdisease, ulcerative colitis) include substances that suppress cytokineproduction, down-regulate or suppress self-antigen expression, or maskthe MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);non-steroidal antiinflammatory drugs (NSAIDs); ganciclovir; tacrolimus;lucocorticoids such as Cortisol or aldosterone; anti-inflammatory agentssuch as a cyclooxygenase inhibitor; a 5-lipoxygenase inhibitor; or aleukotriene receptor antagonist; purine antagonists such as azathioprineor mycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporine; 6-mercaptopurine; steroids such as corticosteroids orglucocorticosteroids or glucocorticoid analogs, e.g., prednisone,methylprednisolone, including SOLU-MEDROL®, methylprednisolone sodiumsuccinate, and dexamethasone; dihydrofolate reductase inhibitors such asmethotrexate (oral or subcutaneous); anti-malarial agents such aschloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokineor cytokine receptor antibodies or antagonists includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosisfactor(TNF)-alpha antibodies (infliximab (REMICADE®) or adalimumab),anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies,anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies,and anti-interleukin-6 (IL-6) receptor antibodies and antagonists;anti-LFA-1 antibodies, including anti-CD 1 1a and anti-CD 18 antibodies;anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, anti-CD3 or anti-CD4/CD4a antibodies; soluble peptidecontaining a LFA-3 binding domain (WO 90/08187 published Jul. 26, 1990);streptokinase; transforming growth factor-beta (TGF-beta);streptodomase; RNA or DNA from the host; FK506; RS-61443; chlorambucil;deoxyspergualin; rapamycin; T-cell receptor (Cohen et al, U.S. Pat. No.5,114,721); T-cell receptor fragments (Offner et al, Science, 251:430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO91/01133); BAFF antagonists such as BAFF or BR3 antibodies orimmunoadhesins and zTNF4 antagonists (for review, see Mackay and Mackay,Trends Immunol, 23: 113-5 (2002) and see also definition below);biologic agents that interfere with T cell helper signals, such asanti-CD40 receptor or anti-CD40 ligand (CD 154), including blockingantibodies to CD40-CD40 ligand. (e.g., Durie et al, Science, 261:1328-30 (1993); Mohan et al, J. Immunol, 154: 1470-80 (1995)) andCTLA4-Ig (Finck et al, Science, 265: 1225-7 (1994)); and T-cell receptorantibodies (EP 340,109) such as T10B9. Non-limiting examples of adjunctagents also include the following: budenoside; epidermal growth factor;aminosalicylates; metronidazole; mesalamine; olsalazine; balsalazide;antioxidants; thromboxane inhibitors; IL-1 receptor antagonists;anti-IL-1 monoclonal antibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; TNF antagonists; IL-4, IL-10, IL-13and/or TGFβ cytokines or agonists thereof (e.g., agonist antibodies);IL-11; glucuronide- or dextran-conjugated prodrugs of prednisolone,dexamethasone or budesonide; ICAM-I antisense phosphorothioateoligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); solublecomplement receptor 1 (TP10; T Cell Sciences, Inc.); slow-releasemesalazine; antagonists of platelet activating factor (PAF);ciprofloxacin; and lignocaine. Examples of agents for UC aresulfasalazine and related salicylate-containing drugs for mild cases andcorticosteroid drugs in severe cases. Topical administration of eithersalicylates or corticosteroids is sometimes effective, particularly whenthe disease is limited to the distal bowel, and is associated withdecreased side effects compared with systemic use. Supportive measuressuch as administration of iron and antidiarrheal agents are sometimesindicated. Azathioprine, 6-mercaptopurine and methotrexate are sometimesalso prescribed for use in refractory corticosteroid-dependent cases.

In other embodiments, a TLR agonist as described herein can beadministered with one or more of: a CHST15 inhibitor, a IL-6 receptorinhibitor, a TNF inhibitor, an integrin inhibitor, a JAK inhibitor, aSMAD7 inhibitor, a IL-13 inhibitor, an IL-1 receptor inhibitor, anIL-12/IL-23 inhibitor, an immunosuppressant, a live biotherapeutic suchas a stem cell, IL-10 or an IL-10 agonist, copaxone, a CD40 inhibitor,an S1P-inhibitor, or a chemokine/chemokine receptor inhibitor. In otherembodiments, a TLR agonist as described herein can be administered witha vitamin C infusion, one or more corticosteroids, and optionallythiamine.

In some embodiments, the methods disclosed herein comprise administering(i) the TLR agonist as disclosed herein, and (ii) a second agent orally,intravenously or subcutaneously, wherein the second agent in (ii) is thesame TLR agonist in (i); a different TLR agonist; or an agent having adifferent biological target from the TLR agonist.

In some embodiments, the methods disclosed herein comprise administering(i) the TLR agonist in the manner disclosed herein, and (ii) a secondagent orally, intravenously or subcutaneously, wherein the second agentin (ii) is an agent suitable for treating an inflammatory bowel disease.

In some embodiments, the TLR agonist is administered prior to the secondagent. In some embodiments, the TLR agonist is administered after thesecond agent. In some embodiments, the TLR agonist and the second agentare administered substantially at the same time. In some embodiments,the TLR agonist is delivered prior to the second agent. In someembodiments, the TLR agonist is delivered after the second agent. Insome embodiments, the TLR agonist and the second agent are deliveredsubstantially at the same time.

In some embodiments, the second agent is an agent suitable for thetreatment of a disease of the gastrointestinal tract. In someembodiments, the second agent is an agent suitable for the treatment ofan inflammatory bowel disease. In some embodiments, the second agent isadministered intravenously. In some embodiments, the second agent isadministered subcutaneously. In some embodiments, the second agent ismethotrexate.

In some embodiments, delivery of the TLR agonist to the location, suchas delivery to the location by mucosal contact, results in systemicimmunogenicity levels at or below systemic immunogenicity levelsresulting from administration of the TLR agonist systemically. In someembodiments comprising administering the TLR agonist in the mannerdisclosed herein and a second agent systemically, delivery of the TLRagonist to the location, such as delivery to the location by mucosalcontact, results in systemic immunogenicity levels at or below systemicimmunogenicity levels resulting from administration of the TLR agonistsystemically and the second agent systemically. In some embodiments, themethod comprises administering the TLR agonist in the manner disclosedherein and a second agent, wherein the amount of the second agent isless than the amount of the second agent when the TLR agonist and thesecond agent are both administered systemically. In some aspects ofthese embodiments, the second agent is a TLR agonist.

In some embodiments, the method comprises administering the TLR agonistin the manner disclosed herein and does not comprise administering asecond agent.

EXAMPLES Example 1—Preclinical Murine Colitis Model

Experimental Induction of Colitis

Colitis is experimentally induced to mice via the dextran sulfate sodium(DSS)-induced colitis model. This model is widely used because of itssimplicity and many similarities with human ulcerative colitis. Briefly,mice are subjected to DSS via cecal catheterization, which is thought tobe directly toxic to colonic epithelial cells of the basal crypts, forseveral days until colitis is induced.

Groups

Mice are allocated to one of seven cohorts, depending on the agent thatis administered:

-   -   1. Control (no agent)    -   2. Adalimumab (2.5 mg/kg)    -   3. Adalimumab (5 mg/kg)    -   4. Adalimumab (10 mg/kg)

The control or agent is applied to a damaged mucosal surface of thebowel via administration through a cecal catheter at the dose levelsdescribed above.

Additionally, for each cohort, the animals are separated into twogroups. One group receives a single dose of the control or agent on day10 or 12. The other group receives daily (or similar) dosing of thecontrol or agent.

Analysis

For each animal, efficacy is determined (e.g., by endoscopy, histology,etc.), and cytotoxic T-cell levels are determined in blood, feces, andtissue (tissue levels are determined after animal sacrifice). For tissuesamples, levels HER2 are additionally determined, and the level ofcytotoxic T cells is normalized to the level of HER2. Additionally,other cytokine levels are determined in tissue (e.g., phospho STAT 1,STAT 3 and STAT 5), in plasma (e.g., VEGF, VCAM, ICAM, IL-6), or both.

Pharmacokinetics are determined both systemically (e.g., in the plasma)and locally (e.g., in colon tissue). For systemic pharmacokineticanalysis, blood and/or feces is collected from the animals at one ormore timepoints after administration (e.g., plasma samples are collectedat 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and/or 8 hoursafter administration). Local/colon tissue samples are collected onceafter animal sacrifice.

Example 2a—Development of Preclinical Porcine Colitis Model

Experimental Induction of Colitis

Female swine weighing approximately 35 to 45 kg at study start arefasted at least 24 hours prior to intra-rectal administration oftrinitrobenzene sulfonic acid (TNBS). Animals are lightly anesthetizedduring the dosing and endoscopy procedure. An enema to clean the colonis used, if necessary. One animal is administered 40 ml of 100% EtOHmixed with 5 grams of TNBS diluted in 10 ml of water via an enema usinga ball-tipped catheter. The enema is deposited in the proximal portionof the descending colon just past the bend of the transverse colon. TheTNBS is retained at the dose site for 12 minutes by use of two Foleycatheters with 60-ml balloons placed in the mid-section of thedescending colon below the dose site. A second animal is similarlytreated, but with a solution containing 10 grams of TNBS. An Endoscopeis employed to positively identify the dose site in both animals priorto TNBS administration. Dosing and endoscopy are performed by aveterinary surgeon

Seven (7) days after TNBS administration, after light anesthesia, thedose site and mucosal tissues above and below the dose site areevaluated by the veterinary surgeon using an endoscope. Pinch Biopsiesare obtained necessary, as determined by the surgeon. Based on theendoscopy findings, the animals may be euthanized for tissue collectionon that day, or may proceed on study pending the results of subsequentendoscopy exams for 1 to 4 more days. Macroscopic and microscopicalterations of colonic architecture, possible necrosis, thickening ofthe colon, and substantial histologic changes are observed at the properTNBS dose.

Clinical signs (e.g., ill health, behavioral changes, etc.) are recordedat least daily during acclimation and throughout the study. Additionalpen-side observations are conducted twice daily (once-daily onweekends). Body weight is measured for both animals Days 1 and 7 (and onthe day of euthanasia if after Day 7).

On the day of necropsy, the animals are euthanized via injection of aveterinarian-approved euthanasia solution. Immediately after euthanasiain order to avoid autolytic changes, colon tissues are collected,opened, rinsed with saline, and a detailed macroscopic examination ofthe colon is performed to identify macroscopic finings related toTNBS-damage. Photos are taken. Tissue samples are taken from theproximal, mid, and distal transverse colon; the dose site; the distalcolon; the rectum; and the anal canal. Samples are placed into NBF andevaluated by a board certified veterinary pathologist.

Example 2b—Pharmacokinetic/Pharmacodynamic and Bioavailability ofAdalimumab after Topical Application

Groups

Sixteen (16) swine (approximately 35 to 45 kg at study start) areallocated to one of five groups:

-   -   1. Vehicle Control: (3.2 mL saline); intra-rectal; (n=2)    -   2. Treated Control: Adalimumab (40 mg in 3.2 mL saline);        subcutaneous; (n=2)    -   3. Adalimumab (low): Adalimumab (40 mg in 3.2 mL saline);        intra-rectal; (n=4)    -   4. Adalimumab (med): Adalimumab (80 mg in 3.2 mL saline);        intra-rectal; (n=4)    -   5. Adalimumab (high): Adalimumab (160 mg in 3.2 mL saline);        intra-rectal; (n=4)

On Day 0, the test article is applied to a damaged mucosal surface ofthe bowel via intra-rectal administration or subcutaneous injection by aveterinary surgeon at the dose levels and volume described above.

Clinical Observations and Body Weight

Clinical observations are conducted at least once daily. Clinical signs(e.g., ill health, behavioral changes, etc.) are recorded on allappropriate animals at least daily prior to the initiation of experimentand throughout the study until termination. Additional clinicalobservations may be performed if deemed necessary. Animals whose healthcondition warrants further evaluation are examined by a ClinicalVeterinarian. Body weight is measured for all animals Days −6, 0, andafter the last blood collections.

Samples

Blood:

Blood is collected (cephalic, jugular, and/or catheter) into EDTA tubesduring acclimation on Day-7, just prior to dose on Day 0, and 0.5, 1, 2,4, 6, 8, 12, 24, and 48 hours post-dose. The EDTA samples are split intotwo aliquots and one is centrifuged for pharmacokinetic plasma andeither analyzed immediately, or stored frozen (−80° C.) for laterpharmacokinetic analyses. The remaining sample of whole blood is usedfor pharmacodynamic analyses.

Feces:

Feces is collected Day −7, 0 and 0.5, 1, 2, 4, 6, 8, 12, 24 and 48 hourspost-dose, and either analyzed immediately, or flash-frozen on liquidnitrogen and stored frozen at −70° C. pending later analysis of druglevels and inflammatory cytokines.

Tissue:

Immediately after euthanasia in order to avoid autolytic changes, colontissues are collected, opened, rinsed with saline, and a detailedmacroscopic examination of the colon is performed to identifymacroscopic finings related to TNBS-damage. Triplicate samples of normaland damaged tissues are either analyzed immediately, or are flash-frozenon liquid nitrogen and stored frozen at −70° C. pending later analysisof drug concentration, inflammatory cytokines and histology.

Samples are analyzed for adalimumab levels (local mucosal tissue levelsand systemic circulation levels), and for levels of inflammatorycytokines including TNF-alpha.

Terminal Procedures

Animals are euthanized as per the schedule in Table AA, where one animaleach of Vehicle and Treated Control groups is euthanized at 6 and 48hours post-dose, and one animal of each the adalimumab groups areeuthanized at 6, 12, 24 and 48 hours post-dose. Animals are discardedafter the last blood collection unless retained for a subsequent study.

TABLE AA Sample Days Hours General size Dose Route −7 6 −5 −4 −3 −2 1 00.5 1 2 4 6 8 12 24 48 Fast • Food/Water ad libidum oral • • • • • • • •• • • • • • • • Observations clinical observations • • • • • • • • bodyweight • • • • Treatments (groups) TNBS (all animals) intra rectal •1.Vehicle control n = 2 1.6 mL saline intra rectal • (vehicle)euthanized n = 1 n = 1 2. Treated control n = 2 43 mg in sub-cutaneous1.6 mL saline euthanized n = 1 n = 1 3. Adalimumab (low) n = 4 40 mg inintra rectal • 1.6 mL saline euthanized n = 1 n = 1 n = 1 n = 1 4.Adalimumab (med) n = 4 80 mg in intra rectal • 1.6mL saline euthanized n= 1 n = 1 n = 1 n = 1 5. Adalimumab (high) n = 4 160 mg in intra rectal• 1.6 mL saline euthanized n = 1 n = 1 n = 1 n = 1 Adalimumab (required)1200 Samples Blood cephalic, jugular or • • • • • • • • • • • catheterFecal rectal • • • • • • • • • • • Tissue necropsy • • • • •

Example 2c—Pharmacokinetic/Pharmacodynamic and Bioavailability ofAdalimumab after Topical Application

Groups

DSS-induced colitis Yorkshire-Cross Farm Swine (approximately 5-10 kg atstudy start) are allocated to one of five groups:

-   -   1. Vehicle Control: (saline); intra-rectal;    -   2. Treated Control: Adalimumab (13 mg in saline); subcutaneous;    -   3. Adalimumab: Adalimumab (13 mg in saline); intra-rectal;

At t=0, the test article is applied to a damaged mucosal surface of thebowel via intra-rectal administration or subcutaneous injection by aveterinary surgeon at the dose levels and volume described above.

Clinical Observations

Clinical signs (e.g., ill health, behavioral changes, etc.) are recordedon all appropriate animals at least daily prior to the initiation ofexperiment and throughout the study until termination. Additionalclinical observations may be performed if deemed necessary. Animalswhose health condition warrants further evaluation are examined by aClinical Veterinarian.

Samples

Blood:

Blood is collected (cephalic, jugular, and/or catheter) into EDTA tubesduring acclimation on Day −7, just prior to dose on Day 0, and 12 hourspost-dose. The EDTA samples are split into two aliquots and one iscentrifuged for pharmacokinetic plasma and either analyzed immediately,or stored frozen (−80° C.) for later pharmacokinetic analyses. Theremaining sample of whole blood is used for pharmacodynamic analyses.

Feces:

Feces is collected Day −7, 0 and 12 hours post-dose, and either analyzedimmediately, or flash-frozen on liquid nitrogen and stored frozen at−70° C. pending later analysis of drug levels and inflammatorycytokines.

Tissue:

Immediately after euthanasia (12 hours after dosing) in order to avoidautolytic changes, colon tissues are collected, opened, rinsed withsaline, and a detailed macroscopic examination of the colon is performedto identify macroscopic finings related to DSS-damage. Triplicatesamples of normal and damaged tissues are either analyzed immediately,or are flash-frozen on liquid nitrogen and stored frozen at −70° C.pending later analysis of drug concentration, inflammatory cytokines andhistology.

Samples are analyzed for adalimumab levels (local mucosal tissue levelsand systemic circulation levels), and for levels of inflammatorycytokines including TNF-alpha.

Terminal Procedures

Animals are euthanized at 12 hours post-dose.

Example 3. Comparison of Systemic Versus Intracecal Delivery of anAnti-IL-12 Antibody

The objective of this study was to compare the efficacy of an IL-12inhibitor (anti-IL-12 p40; anti-p40 mAb; BioXCell (Cat #: BE0051)), whendosed systemically versus intracecally, to the treat dextran sulfatesodium salt (DSS)-induced colitis in male C57Bl/6 mice.

Materials and Methods

Mice

Normal male C57Bl/6 mice between the ages of 6-8 weeks old, weighing20-24 g, were obtained from Charles River Laboratories. The mice wererandomized into thirteen groups of twelve animals and two groups ofeight animals, and housed in groups of 6-8 per cage, and acclimatizedfor at least three days prior to entering the study. Animal rooms wereset to maintain a minimum of 12 to 15 air changes per hour, with anautomatic timer for a light/dark cycle of 12 hours on/off, and fed withLabdiet 5053 sterile rodent chow, with water administered ad libitum.

Cecal Cannulation

Animals were placed under isoflurane anesthesia, with the cecum exposedvia a midline incision in the abdomen. A small point incision was madein the distal cecum where 1-2 cm of the cannula was inserted. Theincision was closed with a purse string suture using 5-0 silk. Anincision was then made in the left abdominal wall through which thedistal end of the cannula was inserted and pushed subcutaneously to thedorsal aspect of the back. The site was then washed copiously withwarmed saline prior to closing the abdominal wall. A small incision wasalso made in the skin of the back between the shoulder blades, exposingthe tip of the cannula. The cannula was secured in place using suture,wound clips, and tissue glue. All animals received 1 mL of warm sterilesaline (subcutaneous injection) and were monitored closely untilrecovery before returning to their cage. All animals received 0.6 mg/kgBID buprenorphine for the first 3 days, and Baytril® at 10 mg/Kg everyday for the first 5 days post surgery.

Induction of Colitis

Colitis was induced in male C57Bl/6 mice by exposure to 3% DSS drinkingwater (MP Biomedicals #0260110) from Day 0 to Day 5. Fresh DSS/watersolutions were made again on Day 3 and any of the remaining original DSSsolution will be discarded.

Assessment of Colitis

All animals were weighed daily and visually assessed for the presence ofdiarrhea and/or bloody stool at the time of dosing. The mice underwenttwo video endoscopies, one on day 10 and one on day 14, to assesscolitis severity. Images were captured from each animal at the mostsevere region of disease identified during the endoscopy, and assessedusing the rubric demonstrated in Table 1.1. Additionally, stoolconsistency was scored during the endoscopy using this rubric (Table1.2) (0=Normal, well-formed pellet, 1=Loose stool, soft, staying inshape, 2=Loose stool, abnormal form with excess moisture, 3=Watery ordiarrhea, 4=Bloody diarrhea). At necropsy, intestinal contents,peripheral blood, and tissue, and cecum/colon contents were collectedfor analysis.

TABLE 1.1 Endoscopy Scoring Score Description of Endoscopy Score 0Normal 1 Loss of vascularity 2 Loss of vascularity and friability 3Friability and erosions 4 Ulcerations and bleeding

TABLE 1.2 Stool Consistency Score Score Description of Stool Consistency0 Normal, well-formed pellet 1 Loose stool, soft, staying in shape 2Loose stool, abnormal form with excess moisture 3 Watery or diarrhea 4Bloody diarrheaTreatment of Colitis

Mice were treated with anti-IL-12 p40 during the acute phase of colitisdue to its efficacy in the treatment of DSS-induced colitis. The testarticle was dosed at a volume of 0.1 mL/20 g from days 0 to 14.Anti-IL-12 p40 was administered intraperitoneally at a dose of 10 mg/kgevery 3 days, and intracecally at a dose of 10 mg/kg, either every 3days or every day. There was also a lower dose of 1 mg/kg given everyday intracecally. The control groups were not administered drugs, andthe vehicles (sterile PBS) were administered the placebo drugintraperitoneally and intracecally every day. These drugs were givenfrom days 5-14, which is 9 days of administration. A more detailedexplanation of dosing and groups can be seen in Table 1.3.

TABLE 1.3 Groups of Animals # of Dose Group Ani- Cecal Treat- (mg/Dosing # mals DSS Cannula ment kg) Route Schedule 1  8 — NO — — — —males 2  8 — YES — — — — males 3 12 3% DSS NO Vehicle — PO QD males (day0-5) day 0-14 4 12 3% DSS YES Vehicle — IC QD males (day 0-5) day 0-14 512 3% DSS NO Anti-p40 10 IP Q3 males (day 0-5) 0,3,6,9,12 6 12 3% DSSYES Anti-p40 10 IC Q3 males (day 0-5) 0,3,6,9,12 7 12 3% DSS YESAnti-p40 10 IC QD males (day 0-5) day 0-14 8 12 3% DSS YES Anti-p40  1IC QD males (day 0-5) day 0-14Sample Collection

Intestinal contents, peripheral blood, and tissue were collected atsacrifice on day 14, as follows: at the end of each study period, micewere euthanized by CO₂ inhalation immediately following endoscopy on day14. The blood was collected via cardiac puncture into K₂EDTA-coatedtubes and centrifuged at 4000×g for 10 minutes. The blood cell pelletwas retained and snapped frozen. The resulting plasma was then splitinto two separate cryotubes, with 100 μL in one tube and the remainderin the second. Plasma and cell pellet were also collected, flash frozen,and stored at −80 degrees Celsius.

The cecum and colon were removed from each animal and contents werecollected, weighed, and snap frozen in separate cryovials. The colon wasexcised, rinsed, measured, weighed, and then trimmed to 6 cm in lengthand divided into 5 pieces. The most proximal 1 cm of colon was snappedfrozen for subsequent bioanalysis of test article levels. Of theremaining 5 cm of colon, the most distal and proximal 1.5-cm sectionswas placed in formalin for 24 hours then transferred to 70% ethanol forsubsequent histological evaluation. The middle 2-cm portion was bisectedlongitudinally and placed into two separate cryotubes, weighed, and snapfrozen in liquid nitrogen.

Results

The data in FIG. 30 show that the DSS mice that were intracecallyadministered an anti-IL-12 p40 (IgG2A) antibody had decreased weightloss as compared to DSS mice that were intraperitoneally administeredthe anti-IL-12 p40 antibody.

The data in FIG. 31 show that the plasma concentration of the anti-IL-12p40 antibody was decreased in DSS mice that were intracecallyadministered the anti-IL-12 p40 antibody as compared to DSS mice thatwere intraperitoneally administered the anti-IL-12 p40 antibody. Thedata in FIG. 32 show that the cecum and colon concentration of theanti-IL-12 p40 antibody is increased in DSS mice that were intracecallyadministered the anti-IL-12 p40 antibody as compared to the DSS micethat were intraperitoneally administered the anti-IL-12 p40 antibody.

The data in FIGS. 33 and 34 show that the anti-IL-12 p40 antibody isable to penetrate colon tissues (the lumen superficial, lamina propria,submucosa, and tunica muscularis/serosa) in DSS mice intracecallyadministered the anti-IL-12 p40 antibody, while the anti-IL-12 p40antibody did not detectably penetrate the colon tissues of DSS miceintraperitoneally administered the anti-IL-12 p40 antibody. The data inFIG. 35 also show that the ratio of the concentration of anti-IL-12 p40antibody in colon tissue to the concentration of the anti-IL-12 p40antibody in plasma is increased in DSS mice intracecally administeredthe anti-IL-12 p40 antibody as compared to the ratio in DSS miceintraperitoneally administered the anti-IL-12 p40 antibody.

The data in FIG. 36 show that the concentration of IL-1β in colon tissueis decreased in DSS mice intracecally administered the anti-IL-12 p40antibody as compared to the concentration of IL-1β in colon tissue inDSS mice intraperitoneally administered the anti-IL-12 p40 antibody. Thedata in FIG. 37 show that the concentration of IL-6 in colon tissue isdecreased in DSS mice intracecally administered the anti-IL-12 p40antibody as compared to the concentration of IL-6 in colon tissue in DSSmice intraperitoneally administered the anti-IL-12 p40 antibody. Thedata in FIG. 38 show that the concentration of IL-17A in colon tissue isdecreased in DSS mice intracecally administered the anti-IL-12 p40antibody as compared to the concentration of IL-17A in colon tissue inDSS mice intraperitoneally administered the anti-IL-12 p40 antibody.

No significant differences in clinical observations orgastrointestinal-specific adverse effects, including stool consistencyand/or bloody stool, were observed due to cannulation or intra-cecaltreatments when compared with vehicle. No toxicity resulting from thetreatments was reported. A significant reduction in body weight-loss(AUC) was found in groups treated with anti-IL-12 p40 antibody (10 mg/kgand 1 mg/kg, QD) via intra-cecal delivery when compared with vehiclecontrol and intraperitoneal delivery (10 mg/kg, Q3D). Theimmunohistochemistry staining in anti-IL-12 p40 antibody (10 mg/kg, QD)treatment groups showed penetration of the antibody in all layers ofcolon tissue, including lumen mucosa, lamina propria, submucosa, tunicamuscularis, via intra-cecal delivery. The distribution of anti-IL-12 p40antibody was found in all segments of the colon, however, higher levelswere detected in the proximal region. A significantly higher meanconcentration of anti-IL-12 p40 antibody was found in thegastrointestinal contents and colon tissues when delivered viaintra-cecal administration (Anti-p40: 10 mg/kg and 1 mg/kg, QD) comparedwith intraperitoneal administration (anti-p40: 10 mg/kg, Q3D). The bloodlevel of anti-IL-12 p40 antibody was significantly higher when deliveredvia intraperitoneal administration (Q3D) as compared to intra-cecaladministration (Q3D & QD). The concentrations of inflammatory cytokines,including IL-1β, IL-6, and IL-17, were significantly reduced byanti-IL-12 p40 antibody (10 mg/kg, QD) treatment when delivered viaintra-cecal administration as compared to vehicle controls.

In sum, these data show that the compositions and devices providedherein can suppress the local immune response in the intestine, whilehaving less of a suppressive effect on the systemic immune response ofan animal. These data also suggest that the presently claimedcompositions and devices will provide for treatment of colitis and otherpro-inflammatory disorders of the intestine.

Example 4. Comparison of Systemic Versus Intracecal Delivery of anAnti-Integrin α4β7 Antibody

The objective of this study was to compare the efficacy of an integrininhibitor (anti-integrin α4β7; anti-LPAM1; DATK-32 mAb; BioXCell (Cat #:BE0034)) when dosed systemically versus intracecally for treatingdextran sulfate sodium salt (DSS)-induced colitis in male C57Bl/6 mice.

Materials and Methods

Mice

Normal male C57Bl/6 mice between the ages of 6-8 weeks old, weighing20-24 g, were obtained from Charles River Laboratories. The mice wererandomized into thirteen groups of twelve animals and two groups ofeight animals, and housed in groups of 6-8 per cage, and acclimatizedfor at least three days prior to entering the study. Animal rooms wereset to maintain a minimum of 12 to 15 air changes per hour, with anautomatic timer for a light/dark cycle of 12 hours on/off, and fed withLabdiet 5053 sterile rodent chow, with water administered ad libitum.

Cecal Cannulation

The animals were placed under isoflurane anesthesia, with the cecumexposed via a midline incision in the abdomen. A small point incisionwas made in the distal cecum where 1-2 cm of the cannula was inserted.The incision was closed with a purse string suture using 5-0 silk. Anincision was then made in the left abdominal wall through which thedistal end of the cannula was inserted and pushed subcutaneously to thedorsal aspect of the back. The site was then washed copiously withwarmed saline prior to closing the abdominal wall. A small incision wasalso made in the skin of the back between the shoulder blades, exposingthe tip of the cannula. The cannula was secured in place using suture,wound clips, and tissue glue. All animals received 1 mL of warm sterilesaline (subcutaneous injection) and were monitored closely untilrecovery before returning to their cage. All animals received 0.6 mg/kgBID buprenorphine for the first 3 days, and Baytril® at 10 mg/Kg everyday for the first 5 days post-surgery.

Induction of Colitis

Colitis was induced in male C57Bl/6 mice by exposure to 3% DSS drinkingwater (MP Biomedicals #0260110) from day 0 to day 5. Fresh DSS/watersolutions were made again on day 3 and any of the remaining original DSSsolution will be discarded.

Assessment of Colitis

All animals were weighed daily and visually assessed for the presence ofdiarrhea and/or bloody stool at the time of dosing. Mice underwent twovideo endoscopies, one on day 10 and one on day 14, to assess colitisseverity. Images were captured from each animal at the most severeregion of disease identified during the endoscopy, and assessed usingthe rubric demonstrated in Table 2.1. Additionally, stool consistencywas scored during the endoscopy using this rubric (Table 2.2) (0=Normal,well-formed pellet, 1=Loose stool, soft, staying in shape, 2=Loosestool, abnormal form with excess moisture, 3=Watery or diarrhea,4=Bloody diarrhea). At necropsy, intestinal contents, peripheral bloodand tissue, and cecum/colon contents were collected for analysis.

TABLE 2.1 Endoscopy Score Score Description of Endoscopy Score 0 Normal1 Loss of vascularity 2 Loss of vascularity and friability 3 Friabilityand erosions 4 Ulcerations and bleeding

TABLE 2.2 Stool Consistency Score Score Description of Stool Consistency0 Normal, well-formed pellet 1 Loose stool, soft, staying in shape 2Loose stool, abnormal form with excess moisture 3 Watery or diarrhea 4Bloody diarrheaTreatment of Colitis

Mice were treated with DATK32 during the acute phase of colitis due toits efficacy in the treatment of DSS-induced colitis. The test articlewas dosed at a volume of 0.1 mL/20 g from days 0 to 14. DATK32 wasadministered intraperitoneally at a dose of 25 mg/kg every 3 days, andintracecally at a dose of 25 mg/kg, either every 3 days or every day.There was also a lower dose of 5 mg/kg given every day intracecally. Thecontrol groups were not administered drugs, and the vehicle (sterilePBS) was administered as the placebo drug intraperitoneally andintracecally every day. These drugs were given from days 5-14, which is9 days of administration. A more detailed explanation of dosing andgroups can be seen in Table 2.3.

TABLE 2.3 Groups of Mice # of Dose Group Ani- Cecal Treat- (mg/ Dosing #mals DSS Cannula ment kg) Route Schedule 1  8 — NO — — — — males 2  8 —YES — — — — males 3 12 3% DSS NO Vehicle — PO QD males (day 0-5) day0-14 4 12 3% DSS YES Vehicle — IC QD males (day 0-5) day 0-14 9 12 3%DSS NO DATK32 25 IP Q3 males (day 0-5) 0,3,6,9,12 10 12 3% DSS YESDATK32 25 IC Q3 males (day 0-5) 0,3,6,9,12 11 12 3% DSS YES DATK32 25 ICQD males (day 0-5) day 0-14 12 12 3% DSS YES DATK32  5 IC QD males (day0-5) day 0-14Sample Collection

Intestinal contents, peripheral blood, and tissue were collected atsacrifice on day 14, as follows: at the end of each study period, micewere euthanized by CO₂ inhalation immediately following endoscopy on day14. The blood was collected via cardiac puncture into K₂EDTA-coatedtubes and centrifuged at 4000×g for 10 minutes. The blood cell pelletwas retained and snapped frozen. The resulting plasma was then splitinto two separate cryotubes, with 100 μL in one tube and the remainderin the second. Plasma and the cell pellet were also collected, flashfrozen, and stored at −80 degrees Celsius. An ELISA was used todetermine the level of rat IgG2A.

The cecum and colon were removed from each animal and contents werecollected, weighed, and snap frozen in separate cryovials. The colon wasexcised, rinsed, measured, weighed, and then trimmed to 6 cm in lengthand divided into 5 pieces. The most proximal 1 cm of colon was snappedfrozen for subsequent bioanalysis of anti-DATK32 levels. Of theremaining 5 cm of colon, the most distal and proximal 1.5-cm sectionswas placed in formalin for 24 hours then transferred to 70% ethanol forsubsequent histological evaluation. The middle 2-cm portion was bisectedlongitudinally and placed into two separate cryotubes, weighed, and snapfrozen in liquid nitrogen.

There was an additional collection of 100 μL of whole blood from allanimals and processed for FACS analysis of α4 and β7 expression onT-helper memory cells. Tissue and blood were immediately placed in FACSbuffer (1×PBS containing 2.5% fetal calf serum) and analyzed using thefollowing antibody panel (Table 2.4).

TABLE 2.4 Fluorophore Labelled Antibodies Used in FACS Analysis AntibodyTarget Flurochrome Purpose CD4 APC-Vio770 Defines T-Helper Cells CD44VioBlue Memory/Naive Discrimination CD45RB FITC Memory/NaiveDiscrimination α4 APC Defines T-helper memory subset of interest β7 PEDefines T-helper memory subset of interest CD16/32 — Fc BlockResults

The data in FIG. 39 show decreased weight loss in DSS mice intracecallyadministered DATK antibody as compared to DSS mice that wereintraperitoneally administered the DATK antibody. The data in FIG. 40show that DSS mice intracecally administered DATK antibody have adecreased plasma concentration of DATK antibody as compared to DSS micethat were intraperitoneally administered DATK antibody. The data inFIGS. 41 and 42 show that DSS mice intracecally administered DATKantibody have an increased concentration of DATK antibody in the cecumand colon content as compared to DSS mice intraperitoneally administeredDATK antibody. The data in FIGS. 43 and 44 show that DSS miceintracecally administered DATK antibody have an increased concentrationof DATK antibody in colon tissue as compared to DSS miceintraperitoneally administered DATK antibody. The data in FIGS. 45 and46 show an increased level of penetration of DATK antibody into colontissue in DSS mice intracecally administered the DATK antibody ascompared to an intracecal vehicle control (PBS). The data in FIG. 47show that DSS mice intracecally administered DATK antibody have anincreased ratio of the concentration of DATK antibody in colon tissue tothe plasma concentration of the DATK antibody, as compared to the sameratio in DSS mice intraperitoneally administered the DATK antibody.

The data in FIG. 48 show that DSS mice intracecally administered theDATK antibody have an increased percentage of blood Th memory cells ascompared to DSS mice intraperitoneally administered the DATK antibody.

No significant differences in clinical observations orgastrointestinal-specific adverse effects, including stool consistencyand/or bloody stool, were observed due to cannulation or intra-cecaltreatments when compared with vehicle. No toxicity resulting from thetreatments was reported. A significant reduction in body weight-loss wasalso found with DATK32 (5 mg/kg, QD) treatment (IC) when compared tovehicle control at the endpoint (day 14). The immunohistochemistrystaining in DATK32 (25 mg/kg, QD) treatment groups showed penetration ofDATK32 in all layers of colon tissue, including lumen mucosa, laminapropria, submucosa, tunica muscularis, via intra-cecal delivery. Thedistribution of DATK32 was found in all segments of the colon, however,higher levels were detected in the proximal region. A significantlyhigher mean concentration of DATK32 was found in gastrointestinalcontents and colon tissues when delivered via intra-cecal administration(DATK32: 25 mg/kg and 5 mg/kg, QD) as compared to intraperitonealadministration (DATK32: 25 mg/kg, Q3D). The blood level of DATK32 wassignificantly higher when delivered via intraperitoneal administration(Q3D) as compared to intra-cecal administration (Q3D & QD). Thepharmacokinetics of DATK32 (25 mg/kg, QD) showed significantly highermean concentrations of DATK32 when delivered via intra-cecaladministration at 1, 2, and 4 h post-dose in the gastrointestinalcontents, and 1, 2, 4 and 24 h in colon tissue as compared with the meanconcentrations of DATK32 following intraperitoneal administration. Themean number of gut-homing T cells (Th memory cells) was significantlyhigher in the blood of groups treated with DATK32 via intra-cecaladministration (QD 25 mg/kg and QD 5 mg/kg) as compared to the groupstreated with DATK32 via intraperitoneal administration (Q3D 25 mg/kg).The mean number of Th memory cells was significantly lower in thePeyer's Patches of groups treated with DATK32 via intra-cecaladministration (QD 25 mg/kg and 5 mg/kg) as compared to the groupstreated with DATK32 via intraperitoneal administration (Q3D 25 mg/kg).The mean number of Th memory cells in mesenteric lymph nodes (MLN) wassignificantly lower in groups treated with DATK32 via intra-cecaladministration (QD and Q3D 25 mg/kg and QD 5 mg/kg) as compared to thegroups treated with DATK32 via intraperitoneal administration (Q3D 25mg/kg).

In sum, these data show that the compositions and devices providedherein can suppress the local immune response in the intestine, whilehaving less of a suppressive effect on the systemic immune response ofan animal. These data also show that the release of DATK-32 antibody inthe colon can result in a suppression of leukocyte recruitment and mayprovide for the treatment of colitis and other pro-inflammatory diseasesof the intestine.

Example 5. An Assessment of DATK32 Bio-Distribution Following IntracecalAdministration in Male C57Bl/6 Mice

The objective of this study is to assess DATK32 bio-distribution whendosed intracecally in male C57Bl/6 mice. A minimum of 10 days prior tothe start of the experiment a cohort of animals will undergo surgicalimplantation of a cecal cannula. A sufficient number of animals willundergo implantation to allow for 24 cannulated animals to be enrolledin the main study (e.g., 31 animals). Animals were dosed with vehicle ortest article via intracecal injection (IC) on Day 0 as indicated inTable 3. Animals from all groups were sacrificed for terminal samplecollection three hours following test article administration.

Materials and Methods

Mice

Normal male C57Bl/6 mice between the ages of 6-8 weeks old, weighing20-24 g, were obtained from Charles River Laboratories. The mice wererandomized into two groups of twelve animals, and housed in groups of 12per cage, and acclimatized for at least three days prior to entering thestudy. Animal rooms were set to maintain a minimum of 12 to 15 airchanges per hour, with an automatic timer for a light/dark cycle of 12hours on/off, and fed with Labdiet 5053 sterile rodent chow, with wateradministered ad libitum.

Cecal Cannulation

The animals were placed under isoflurane anesthesia, with the cecumexposed via a midline incision in the abdomen. A small point incisionwas made in the distal cecum where 1-2 cm of the cannula was inserted.The incision was closed with a purse string suture using 5-0 silk. Anincision was then made in the left abdominal wall through which thedistal end of the cannula was inserted and pushed subcutaneously to thedorsal aspect of the back. The site was then washed copiously withwarmed saline prior to closing the abdominal wall. A small incision wasalso made in the skin of the back between the shoulder blades, exposingthe tip of the cannula. The cannula was secured in place using suture,wound clips, and tissue glue. All animals received 1 mL of warm sterilesaline (subcutaneous injection) and were monitored closely untilrecovery before returning to their cage. All animals received 0.6 mg/kgBID buprenorphine for the first 3 days, and Baytril® at 10 mg/Kg everyday for the first 5 days post-surgery.

Dosing

Animals were dosed IC at a volume of 0.075 mL/animal on Days 0 asindicated in Table 3.

Sacrifice

All animals were euthanized by CO₂ inhalation three hours after dosingon Day 0.

Sample Collection

Terminal blood was collected and prepared for plasma using K₂EDTA as theanti-coagulant. The plasma will be split into two cryotubes, with 50 μLin one tube (PK analysis) and the remainder in another (other). Bothsamples were flash-frozen in liquid nitrogen. Plasma was stored at −80°C. for downstream analysis. Mesenteric lymph nodes (mLN) were collected,weighed, and flash-frozen in liquid nitrogen. Mesenteric lymph nodeswere stored at −80° C. for downstream analysis. The small intestine wasexcised and rinsed, and the most distal 1 cm of ilium was dissected,weighed, and flash-frozen in liquid nitrogen. The samples were stored at−80° C. for downstream analysis. The cecum and colon were removed fromeach animal and contents collected, weighed, and snap frozen in separatecryovials. The samples were stored at −80° C. for downstream analysis.The colon was rinsed, and the most proximal 1 cm of colon was weighedand flash-frozen in liquid nitrogen. The snap frozen tissues were storedat −80° C.

TABLE 3 Study Design N^(o) Group Animals Treatment Route ScheduleTerminal Collections Day 0 1 12 Vehicle (PBS) IC Day 0** Blood (plasma)Small 2 12 DATK32 intestine mLN (625 μg)* Colon Colon Contents CecumContents *Per mouse. TA was administered in 0.075 mL/animal. DATK32 wasdelivered in sterile PBS. **Animals were dosed on Day 0 and collectionswere performed 3 hours later.Results

The data in FIGS. 63A-F show no significant differences in clinicalobservations. No gastrointestinal-specific or adverse effects were foundin the group administered DATK32 via intra-cecal administration ascompared to the group administered a vehicle control. No toxicityresulting from the treatments was reported. The level of DATK32 in thegroup intra-cecally administered DATK32 was significantly higher incecum and colon content, and colon tissue compared to the groupadministered a vehicle control at 3 h post-dose. A small amount ofDATK32 was also detected in plasma, small intestine, and mesentericlymph node in the group intra-cecally administered DATK32.

Example 6. Pharmacokinectics/Pharmacodynamics and Bioavailability ofAdalimumab when Applied to a TNBS-Damaged Mucosal Surface (InducedColitis) in Swine

The purpose of this non-Good Laboratory Practice (GLP) study was toexplore the PK/PD, and bioavailability of adalimumab when applied to aTNBS-damaged mucosal surface (induced colitis) in Yorkshire-Cross farmswine, and to determine an appropriate dose and frequency for studieswhere a drug will be delivered by the ingestible device system. Theingestible device system will be capable of delivering a TNF inhibitor(adalimumab) topically and locally to damaged mucosa in human patientswith inflammatory bowel disease (IBD). The TNBS-induced colitis modelwas validated when a single administration on Day 1 of 40 mL of 100%ethanol (EtOH) mixed with 5 grams of TNBS diluted in 10 mL of water viaan enema using a rubber catheter resulted in the intended reproducibleinduction of damaged mucosal surface (induced colitis) inYorkshire-Cross farm swine.

This study investigated whether topical delivery of adalimumab wouldresult in increased local mucosal tissue levels with limited drugreaching systemic circulation, as compared to subcutaneousadministration; whether local mucosal tissue levels of drug would begreater in damaged tissues when compared to normal tissues; whetherincreasing the dose of drug would result in increased mucosal tissuelevels in local and distal TNBS-damaged tissues; and whether topicaldelivery of adalimumab would result in reductions in inflammatorycytokines such as TNF-α in damaged tissues, feces, and possibly blood.

All animals were subjected to intra-rectal administration oftrinitrobenzene sulfonic acid (TNBS) to induce chronic colitis on day−2. All animals were fasted prior to colitis induction. Bedding wasremoved and replaced with rubber mats on day −3 to prevent ingestion ofstraw bedding material. The dose was 40 mL of 100% EtOH mixed with 5grams of TNBS diluted in 10 mL of water, then instilled into the colonintra-rectally using a flexible gavage tube by a veterinary surgeon(deposited in a 10-cm portion of the distal colon and proximal rectum,and retained for 12 minutes by use of two Foley catheters with 60-mLballoons). Approximately 3 days after induction, macroscopic andmicroscopic alterations of colonic architecture were apparent: somenecrosis, thickening of the colon, and substantial histologic changeswere observed (FIGS. 49 and 50). The study employed 15 female swine(approximately 35 to 45 kg at study start) allocated to one of fivegroups. Group 1 employed three animals that were the treated controls.Each animal in Group 1 was administered adalimumab by subcutaneousinjection at 40 mg in 0.8 mL saline. Groups 2, 3, 4, and 5 employed 3animals in each group. Animals in these groups were administeredintra-rectal adalimumab at 40 mg in 0.8 mL saline. The test drug(adalimumab) was administered to all groups on study day 1. Theintra-rectal administrations (Groups 2-5) were applied to damagedmucosal surface of the bowel vial intra-rectal administration by aveterinary surgeon. Blood (EDTA) was collected from all animals(cephalic, jugular, or catheter) on day −3 (n=15), −1 (n=15), and 6(n=15), 12 (n=12), 24 (n=9), and 48 (n=6) hours post-dose (87 bleedstotal). The EDTA samples were split into two aliquots, and one wascentrifuged for PK plasma, and stored frozen (−80° C.) for PK analysesand reporting. Fecal samples were collected for the same time-points (87fecal collections). Fecal samples were flash-frozen in liquid nitrogenand then stored at −80° C. for analysis of drug levels and inflammatorycytokines. Groups 2, 3, 4, and 5 were euthanized and subjected to grossnecropsy and tissue collection 6, 12, 24, and 48 hours post-dose,respectively. Group 1 was similarly euthanized and necropsied 48 hourspost-dose. The animals were euthanized via injection of aveterinarian-approved euthanasia solution as per the schedule.Immediately after euthanasia in order to avoid autolytic changes, colontissues were collected, opened, rinsed with saline, and a detailedmacroscopic examination of the colon were performed to identifymacroscopic findings related to TNBS-damage. Tissue samples were takenfrom the proximal, mid, and distal transverse colon; the dose site; andthe distal colon. Each tissue sample was divided into two approximatehalves; one tissue section was placed into 10% neutral buffered formalin(NBF) and evaluated by a Board certified veterinary pathologist, and theremaining tissue section was flash frozen in liquid nitrogen and storedfrozen at −80° C. Clinical signs (ill health, behavioral changes, etc.)were recorded daily beginning on day −3. Additional pen-sideobservations were conducted once or twice daily. Animals observed to bein ill health were examined by a veterinarian. Body weight was measuredfor all animals on day −3, and prior to scheduled euthanasia. Table 4.1,depicted below, shows the study design.

Materials and Methods

Test Article

Adalimumab (EXEMPTIA™) is a Tumour Necrosis Factor (TNF) inhibitor. Asingle dose was pre-filled in a syringe (40 mg in a volume of 0.8 mL).

TABLE 4.1 Study Design Table Days Hours General Sample size Dose Route−3 −2 −1 1 0.5 1 2 4 6 8 12 24 48 Fast • Food/Water ad libidum oral • •• • • • • • • • • • Observations clinical observations • • • • • • bodyweight • • • • Treatments (groups) TN BS (all animals) intra rectal • 1.Treated control n = 3 40 mg in sub-cutaneous • 0.8 mL saline euthanizedn = 3 2. Adalimumab n = 3 40 mg in intra rectal • 0.8 mL salineeuthanized n = 3 3. Adalimumab n = 3 40 mg in intra rectal • 0.8 mLsaline euthanized n = 3 4. Adalimumab n = 3 40 mg in intra rectal • 0.8mL saline euthanized n = 3 5. Adalimumab n = 3 40 mg in intra rectal •0.8 mL saline euthanized n = 3 Adalimumab (required) 600 Samples PBMCscephalic, jugular or • • • • • catheter Serum cephalic, jugular or • • •• • • • catheter Fecal rectal • • • • • • • Tissue necropsy • • • •Analysis Histopathology 1 location 4 locations inflammed 45 180 H&Enormal 45 180 H&E Blood adalimumab 57 pbl 15 15 12 9 6 TN Fα 87 pbl 1515 15 15 12 9 6 Feces adalimumab 57 pbl 15 15 12 9 6 TN Fα 87 pbl 15 1515 15 12 9 6 Tissue Inflammed adalimumab 45 180 pbl 3 3 3 6 TN Fa 45 180pbl 3 3 3 6 HER2 45 180 pbl 3 3 3 6 Normal adalimumab 45 180 pbl 3 3 3 6TN Fa 45 180 pbl 3 3 3 6 HER2 45 180 pbl 3 3 3 6Results

While subcutaneously administered adalimumab was detected at all timespoints tested in plasma, topically administered adalimumab was barelydetectable in plasma (FIGS. 51 and 52). Both topical delivery andsubcutaneous delivery of adalimumab resulted in reduced levels of TNF-αin colon tissue of TNBS-induced colitis animals, yet topical delivery ofadalimumab was able to achieve a greater reduction in TNF-α levels(FIGS. 53 and 54).

Either subcutaneous or intra-rectal administration of adalimumab waswell tolerated and did not result in death, morbidity, adverse clinicalobservations, or body weight changes. A decreased level of totalTNBS-related inflammatory response was observed by adalimumab treatmentvia intra-rectal administration when applied to the damaged mucosalsurface of the bowel when compared to subcutaneous delivery. Asignificantly higher concentration of adalimumab was measured in bloodfollowing subcutaneous delivery as compared to the blood concentrationfollowing intra-rectal administration. Intra-rectal administration ofadalimumab decreased the total and normalized TNFα concentration overtime (6-48 h) and was more effective at reducing TNFα at the endpoint(48 h) as compared to groups administered adalimumab subcutaneously.

In sum, these data show that the compositions and devices providedherein can suppress the local immune response in the intestine, whilehaving less of a suppressive effect on the systemic immune response ofan animal. For example, these data show that intracecal administrationof adalimumab using a device as described herein can provide for localdelivery of adalimumab to the site of disease, without suppressing thesystemic immune response. These data also show that local administrationof adalimumab using a device as described herein can result in asignificant reduction of the levels of TNFα in diseases animals.

Example 7. Comparison of Systemic Versus Intracecal Delivery ofCyclosporine A

The objective of this study was to compare the efficacy of animmunosuppressant agent (cyclosporine A; CsA) when dosed systemicallyversus intracecally to treat dextran sulfate sodium salt (DSS)-inducedcolitis in male C57Bl/6 mice.

Experimental Design

A minimum of 10 days prior to the start of the experiment a cohort ofanimals underwent surgical implantation of a cecal cannula. A sufficientnumber of animals underwent implantation to allow for 44 cannulatedanimals to be enrolled in the main study (e.g., 76 animals). Colitis wasinduced in 60 male C5Bl/6 mice by exposure to 3% DSS-treated drinkingwater from day 0 to day 5. Two groups of eight additional animals(cannulated and non-cannulated) served as no-disease controls (Groups 1and 2). Animals were dosed with cyclosporine A via intraperitonealinjection (IP), oral gavage (PO), or intracecal injection (IC) from day0 to 14 as indicated in Table 5.1. All animals were weighed daily andassessed visually for the presence of diarrhea and/or bloody stool atthe time of dosing. Mice underwent video endoscopy on days 10 and 14 toassess colitis severity. Images were captured from each animal at themost severe region of disease identified during endoscopy. Additionally,stool consistency was scored during endoscopy using the parametersdefined in Table 5.2. Following endoscopy on day 14, animals from allgroups were sacrificed and underwent terminal sample collection.

Specifically, animals in all treatment groups dosed on day 14 weresacrificed at a pre-dosing time point, or 1, 2, and 4 hours after dosing(n=3/group/time point). Terminal blood was collected via cardiacpuncture and prepared for plasma using K₂EDTA as the anti-coagulant. Theblood cell pellet was retained and snap frozen while the resultingplasma was split into two separate cryotubes, with 100 μL in one tubeand the remainder in the second. Additionally, the cecum and colon wereremoved from all animals; the contents were collected, weighed, and snapfrozen in separate cyrovials. The colon was then rinsed, measured,weighed, and then trimmed to 6 cm in length and divided into fivepieces. The most proximal 1 cm of colon was snap frozen for subsequentbioanalysis of cyclosporine A levels. Of the remaining 5 cm of colon,the most distal and proximal 1.5-cm sections were each placed informalin for 24 hours, then transferred to 70% ethanol for subsequenthistological evaluation. The middle 2-cm portion was bisectedlongitudinally and placed into two separate cryotubes, weighed, and snapfrozen in liquid nitrogen. All plasma and frozen colon tissue werestored at −80° C. for selected end point analysis. For all controlanimals in Groups 1-4, there was an additional collection of 100 μL ofwhole blood from all animals which was then processed for FACS analysisof α4 and β7 expression on Tx memory cells. The details of the study areshown in Table 5.1.

TABLE 5.1 Study Design Group 1 2  3  4 13 14 15 Number Number of 8 8 1212 12 12 12 Animals Cecal NO YES NO YES NO YES YES Cannula DSS N/A N/A3% DSS on Day 0 to Day 5 Treatment none none vehicle vehicle CsA CsA CsADose N/A N/A N/A N/A 10 10  3 (mg/kg) Route N/A N/A N/A N/A PO IC ICDosing N/A N/A QD: Day QD: Day QD: Day QD:Day QD: Day Schedule 0 to 14 0to 14 0 to 14 0 to 14 0 to 14 Endoscopy Days 10 and 14 Schedule*Endpoints Endoscopy, Colon weight/ length, stool score Day 14 TerminalCollection (all groups): Cecal contents, colon contents, plasma, andcolon tissue FACS analysis collection of Groups 1-4: Whole blood for thefollowing FACS panel: CD4, CD44, CD45RB, α4, β7, CD16/32 PK N = 3/ timepoints Sacrifice At pre-dose and 1, 2, and 4 hours post-dosing (Day 14)*Animals were dosed once (QD) on Day 14 and plasma collected (K2EDTA) atpre-dosing, 1, 2, and 4 hours post-dosing from n = 3/group/time point.Each collection was terminal.Experimental ProceduresCecal Cannulation

Animals were placed under isofluorance anesthesia, and the cecum exposedvia a midline incision in the abdomen. A small point incision was madein the distal cecum through which 1-2 cm of the cannula was inserted.The incision was closed with a purse-string suture using 5-0 silk. Anincision was made in the left abdominal wall through which the distalend of the cannula was inserted and pushed subcutaneously to the dorsalaspect of the back. The site was washed copiously with warmed salineprior to closing the abdominal wall. A small incision was made in theskin of the back between the shoulder blades, exposing the tip of thecannula. The cannula was secured in place using suture, wound clips, andtissue glue. All animals received 1 mL of warm sterile saline(subcutaneous injection) and were monitored closely until fullyrecovered before returning to the cage. All animals receivedbuprenorphine at 0.6 mg/kg BID for the first 3 days, and Baytril® at 10mg/kg QD for the first 5 days following surgery.

Disease Induction

Colitis was induced on day 0 via addition of 3% DSS (MP Biomedicals, Cat#0260110) to the drinking water. Fresh DSS/water solutions were made onday 3 and any of the remaining original DSS solution was discarded.

Dosing

Animals were dosed by oral gavage (PO), intraperitoneal injection (IP),or intracecal injection (IC) at a volume of 0.1 mL/20 g on days 0 to 14as indicated in Table 5.1.

Body Weight and Survival

Animals were observed daily (weight, morbidity, survival, presence ofdiarrhea, and/or bloody stool) in order to assess possible differencesamong treatment groups and/or possible toxicity resulting from thetreatments.

Animals Found Dead or Moribund

Animals were monitored on a daily basis and those exhibiting weight lossgreater than 30% were euthanized, and samples were not collected fromthese animals.

Endoscopy

Each mouse underwent video endoscopy on days 10 and 14 using a smallanimal endoscope (Karl Storz Endoskope, Germany) under isofluraneanesthesia. During each endoscopic procedure still images as well asvideo were recorded to evaluate the extent of colitis and the responseto treatment. Additionally, we attempted to capture an image from eachanimal at the most severe region of disease identified during endoscopy.Colitis severity was scored using a 0-4 scale (0=normal; 1=loss ofvascularity; 2=loss of vascularity and friability; 3=friability anderosions; 4=ulcerations and bleeding). Additionally, stool consistencywas scored during endoscopy using the parameters defined in Table 5.2.

TABLE 5.2 Stool Consistency Score Description 0 Normal, well-formedpellet 1 Loose stool, soft, staying in shape 2 Loose stool, abnormalform with excess moisture 3 Watery or diarrhea 4 Bloody diarrheaTissue/Blood for FACS

Tissue and blood were immediately placed in FACS buffer (1×phosphate-buffered saline (PBS) containing 2.5% fetal calf serum (FCS))and analyzed using the antibody panel in Table 5.3.

TABLE 5.3 FACS Antibody Panel Antibody Target Fluorochrome Purpose CD4APC-Vio770 Defines T_(H) cells CD44 VioBlue Memory/Naïve discriminationCD45RB FITC Memory/Naïve discrimination α4 APC Defines T_(H)-memorysubset of interest β7 PE Defines T_(H)-memory subset of interest CD16/32— Fc blockResults

The data in FIG. 55 show a decrease in weight loss is observed in DSSmice intracecally administered cyclosporine A as compared to DSS miceorally administered cyclosporine A. The data in FIG. 56 show a decreasein plasma concentration of cyclosporine A in DSS mice intracecallyadministered cyclosporine A as compared to DSS mice orally administeredcyclosporine A. The data in FIGS. 57-59 show an increased concentrationof cyclosporine A in the colon tissue of DSS mice intracecallyadministered cyclosporine A as compared to the concentration ofcyclosporine A in the colon tissue of DSS mice orally administeredcyclosporine A.

The data in FIG. 60 show that DSS mice intracecally administeredcyclosporine A have an increased concentration of IL-2 in colon tissueas compared to DSS mice orally administered cyclosporine A. The data inFIG. 61 show that DSS mice intracecally administered cyclosporine A havea decreased concentration of IL-6 in colon tissue as compared to DSSmice orally administered cyclosporine A.

In sum, these data show that the compositions and devices providedherein can suppress the local immune response in the intestine, whilehaving less of a suppressive effect on the systemic immune response ofan animal. For example, these data demonstrate that the presentcompositions and devices can be used to release cyclosporine A to theintestine and that this results in a selective immune suppression in thecolon, while having less of an effect on the immune system outside ofthe intesting. These data also suggest that the present compositions anddevices will provide for the treatment of colitis and otherpro-inflammatory disorders of the intestine.

Example 8. Bellows Testing: Drug Stability Bench Test

Experiments were run to evaluate the effects that bellows material wouldhave on the function of a drug used as the dispensable substance. Theexperiments also evaluated the effects on drug function due to shelflife in the bellows.

The adalimumab was loaded into simulated device jigs containing eithertapered silicone bellows or smooth PVC bellows and allowed to incubatefor 4, 24, or 336 hours at room temperature while protected from light.FIG. 64 illustrates the tapered silicone bellows, and FIG. 65illustrates the tapered silicone bellows in the simulated device jig.FIG. 66 illustrates the smooth PVC bellows, and FIG. 67 illustrates thesmooth PVC in the simulated device jig.

The drug was subsequently extracted using the respective dispensingsystems and tested by a competitive inhibition assay. The test methodhas been developed from the literature (Velayudhan et al.,“Demonstration of functional similarity of proposed biosimilar ABP501 toadalimumab” BioDrugs 30:339-351 (2016) and Barbeauet et al.,“Application Note: Screening for inhibitors of TNFα/s TNFR1 Bindingusing AlphaScreen™ Technology”. PerkinElmer Technical Note ASC-016.(2002)), as well as pre-testing development work using control drug andexperiments using the provided AlphaLISA test kits. FIG. 68 demonstratesthe principle of the competition assay performed in the experiment.

The bellows were loaded as follows: aseptically wiped the dispensingport of the simulated ingestible device jig with 70% ethanol; allowed toair dry for one minute; used an adalimumab delivery syringe to load eachset of bellows with 200 μL of drug; took a photo of the loaded device;gently rotated the device such that the drug is allowed to come incontact with all bellows surfaces; protected the bellows from light; andincubate at room temperature for the predetermined time period to allowfull contact of the drug with all bellows' surfaces.

The drug was extracted as follows: after completion of the incubationperiod; the device jig was inverted such that the dispensing port waspositioned over a sterile collection microfuge tube and petri dishbelow; five cubic centimeters of air was drawn into an appropriatesyringe; the lure lock was attached to the device jig; the syringe wasused to gently apply positive pressure to the bellow with air such thatthe drug was recovered in the collection microfuge tube; where possible,a video of drug dispensing was taken; samples were collected from eachbellows type; a control drug sample was collected by directly dispensing200 μL of drug from the commercial dispensing syringe into a sterilemicrofuge tube; the control drug-free sample was collected by directlydispensing 200 μL of PBS using a sterile pipette into a sterilemicrofuge tube; the collected drug was protected from light; and thedrug was diluted over the following dilution range (250, 125, 25, 2.5,0.25, 0.025, 0.0125, 0.0025 μg) in sterile PBS to determine the IC₅₀range of the drug.

To determine any effects storage conditions may have on drug efficacy inthe device, the drug (stored either in the syringe, silicon bellows, PVCbellows) was stored at room temperature while protected from light for24 hours and 72 hours. Samples were then extracted and the steps in thepreceding paragraph were repeated.

The AlphaLISA (LOCI™) test method was used. Human TNFα standard dilutionranges were prepared as described in Table 6.

TABLE 6 [human TNFα] Vol. of Vol. of in standard curve Tube human TNFα(μL) diluent (μL) * (g/mL in 5 μL) (pg/mL in 5 μL) A 10 μL ofreconstituted 90 1E−07 100 000     human TNFα B 60 μL of tube A 1403E−08 30 000    C 60 μL of tube B 120 1E−08 10 000    D 60 μL of tube C140 3E−09 3 000    E 60 μL of tube D 120 1E−09 1 000    F 60 μL of tubeE 140 3E−10 300  G 60 μL of tube F 120 1E−10 100  H 60 μL of tube G 1403E−11 30  I 60 μL of tube H 120 1E−11 10  J 60 μL of tube I 140 3E−12 3K 60 μL of tube J 120 1E−12 1 L 60 μL of tube K 140 3E−13   0.3 M **(background) 0 100 0 0 N ** (background) 0 100 0 0 O ** (background) 0100 0 0 P ** (background) 0 100 0 0

The test was performed as follows: the above standard dilution rangeswere in a separate 96-well plate; to ensure consistent mixing, sampleswere mixed up and down gently with a pipette five times; a 384-well testplate was prepared according to the test layout diagram depicted Table7; five microliters of 10,000 pg/mL TNFα standard from the previouslymade dilution plate was added to each corresponding concentration asshown in Table 6; five microliters of recovered drug (directly from thecommercial syringe (A), from the silicone bellows (B Si), from the PVCbellows (B PVC), or from the PBS control (C) was added into thecorresponding wells described in Table 5; the test plate was incubatedfor one hour at room temperature while protected from light; 10microliters of acceptor beads were added to each previously accessedwell; the wells were incubated for 30 minutes at room temperature whileprotected from light; 10 μL of biotinylated antibody was added to eachpreviously accessed well; the wells were incubated for 15 minutes atroom temperature, while protected from light; the room lights weredarkened and 25 microliters of streptavidin (SA) donor beads were addedto each previously accessed well; the wells were incubated for 30minutes at room temperature while protected from light; the plate wasread in Alpha Mode; and the results were recorded. Upon addition ofreagent(s) in the various steps, each well was pipetted up and downthree times to achieve good mixing.

TABLE 7 1 2 3 4 5 6 7 8 9 10 11 12 A STD2 STD10 250 250 250 250 250 250250 250 250 1.00E+05 10 A A A A A B Si B Si B Si B Si B C STD3 STD11 125125 125 125 125 125 125 125 125 30000 3 A A A A A B Si B Si B Si B Si DE STD4 STD12 25 25 25 25 25 25 25 25 25 10000 1 A A A A A B Si B Si B SiB Si F G STD5 STD13 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 3000 0.333 A A AA A B Si B Si B Si B Si H I STD6 Blank 0.25 0.25 0.25 0.25 0.25 0.250.25 0.25 0.25 1000 0 A A A A A B Si B Si B Si B Si J K STD7 Blank 0.0250.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 300 0 A A A A A B Si BSi B Si B Si L M STD8 Blank 0.013 0.013 0.013 0.013 0.013 0.013 0.0130.013 0.013 100 0 A A A A A B Si B Si B Si B Si N O STD9 Blank 0.0030.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 30 0 A A A A A B Si B SiB Si B Si P 13 14 15 16 17 18 19 20 21 22 23 A 250 250 250 250 250 250250 250 250 250 250 B Si B PVC B PVC B PVC B PVC B PVC C C C C C B C 125125 125 125 125 125 125 125 125 125 125 B Si B PVC B PVC B PVC B PVC BPVC C C C C C D E 25 25 25 25 25 25 25 25 25 25 25 B Si B PVC B PVC BPVC B PVC B PVC C C C C C F G 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.52.5 B Si B PVC B PVC B PVC B PVC B PVC C C C C C H I 0.25 0.25 0.25 0.250.25 0.25 0.25 0.25 0.25 0.25 0.25 B Si B PVC B PVC B PVC B PVC B PVC CC C C C J K 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.0250.025 B Si B PVC B PVC B PVC B PVC B PVC C C C C C L M 0.013 0.013 0.0130.013 0.013 0.013 0.013 0.013 0.013 0.013 0.013 B Si B PVC B PVC B PVC BPVC B PVC C C C C C N O 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.0030.003 0.003 0.003 B Si B PVC B PVC B PVC B PVC B PVC C C C C C P

The data are shown in FIGS. 69-71. The data demonstrate that the bellowsdo not negatively impact the drug function after shelf lives of 4 hours,24 hours, or 336 hours. The IC₅₀ values of the drug dispensed from thebellows were comparable to the IC₅₀ values of the standard dispensationmethod (Table 6). A slight right shift was noted in the bellows curvesafter 24 hours (FIG. 70), but this shift was well within the error barsof the curves. Tables 8-11 represent data of FIGS. 69-71, respectively.Of note, when comparing mean (n=5) RFU data between test articles overthe concentration ranges significant differences (p<0.05) werediscerned. However, these significant differences did not favor eithertest article over time, suggesting that they were not related to theperformance of the material in response to the drug (FIGS. 69-71).

TABLE 8 Needle control Silicone Bellows PVC Bellows (A) (B) (C)  4 Hours0.0174 0.0169 0.0172 24 Hours 0.0180 0.0180 0.0180 336 Hours  0.01440.0159 0.0163

TABLE 9 Statistics (Student's T-test, 2 tailed, non-pair-wise, forsignificance p < 0.05) Needle control (A) Needle control (A) SiliconeDrug (micrograms) vs. Silicone (B) vs. PVC vs. PVC 0.0001 0.911 0.008*0.268 0.0025 0.138 0.390 0.822 0.0125 0.122 0.118 0.771 0.025 0.1430.465 0.020* 0.25 0.591 0.984 0.350 2.5 0.243 0.124 0.169 125 0.8670.688 0.182 250 0.681 0.184 0.108 *p < 0.5 data set

TABLE 10 Statistics (Student's T-test, 2 tailed, non-pair-wise, forsignificance p < 0.05) Needle control (A) Needle control (A) SiliconeDrug (micrograms) vs. Silicone (B) vs. PVC vs. PVC 0.0001 0.132 0.038*0.292 0.0025 0.003* 0.076 0.575 0.0125 0.161 0.022* 0.783 0.025 0.0580.078 0.538 0.25 0.974 0.384 0.198 2.5 0.714 0.080 0.017* 125 0.8730.731 0.269 250 0.798 0.956 0.903 *p < 0.5 data set

TABLE 11 Statistics (Student's T-test, 2 tailed, non-pair-wise, forsignificance p < 0.05) Drug Needle control (A) Needle control (A)Silicone (micrograms) vs. Silicone (B) vs. PVC vs. PVC 0.0001 0.8584490.036847* 0.026444* 0.0025 0.087379 0.280302 0.046767* 0.0125 0.4692820.057232 0.117194 0.025 0.02758* 0.078234 0.373419 0.25 0.4115480.258928 0.400498 2.5 0.368959 0.156574 0.006719* 125 0.948649 0.2467020.463735 250 0.485046 0.128993 0.705543 *p < 0.5 data set

Example 9. A Comparison Study of Systemic Vs Intracecal Delivery ofSMAD7 Bio-Distribution in DSS-Induced Colitis in Male C57Bl/6 Mice

The objective of this study was to compare the efficacy of novel testarticles, e.g., fluorescent SMAD7 antisense oligonucleotides (SMAD7 AS),when dosed systemically versus intracecally in the treatment ofDSS-induced colitis, in male C57Bl/6 mice.

Experimental Design

A minimum of 10 days prior to the start of the experiment a cohort ofanimals underwent surgical implantation of a cecal cannula. A sufficientnumber of animals underwent implantation to allow for 12 cannulatedanimals to be enrolled in the main study (i.e., 16 animals).

Colitis was induced in 12 male C57Bl/6 mice (Groups 4-5) by exposure to3% DSS-treated drinking water from Day 0 to Day 5. Three groups of sixadditional animals per group (n=6 cannulated; n=12 non-cannulated;Groups 1-3) served as no-disease controls (Groups 1-3). All animals wereweighed daily and assessed visually for the presence of diarrhea and/orbloody stool during this time.

Animals were dosed with test-article via oral gavage (PO) or intracecalinjection (IC) once on Day 9 as indicated in Table 12. The animals inGroup 0 were not dosed. The animals in Groups 2 and 4 were dosed PO withSMAD7 antisense. The animals in Groups 3 and 5 were dosed IC with SMAD7antisense.

All animals were euthanized by CO₂ inhalation 12 hours after dosing, onDay 10. Terminal blood was collected into two K₂EDTA tubes and processedfor plasma. Both plasma and pellet samples were snap-frozen in liquidnitrogen and stored at −80° C. Cecum contents were removed and thecontents were split into two aliquots. Both aliquots were weighed andsnap frozen in separate cryovials in liquid nitrogen. The cecum wasexcised and bisected longitudinally; each piece is separately weighedand flash-frozen in liquid nitrogen. The colon contents were removed andthe contents were split into two aliquots. Both aliquots were weighedand snap frozen in separate cryovials in liquid nitrogen. The colon wasthen rinsed, and the most proximal 2 cm of colon was collected. This2-cm portion was bisected longitudinally; each piece was separatelyweighed and flash-frozen in liquid nitrogen. Snap-frozen blood pellet,cecum/colon contents, and tissue samples were used for downstreamfluoremetry or RP-HPLC. The details of the study design are shown inTable 12.

TABLE 12 Study design Terminal No Cecal Colitis Collections GroupAnimals Cannula Induction Treatment Route Schedule Day 10 1 6 NO — — — —Whole blood, 2 6 NO Fluorescently PO QD plasma, cecal 3 6 YES labeled ICDay 9** contents, colon 4 6 NO 3% DSS SMAD7 PO contents, cecal 5 6 YESDays 0-5 antisense IC tissue, colon 50 μg* tissue *Per mouse. TA isadministered in 0.075 mL/animal. **Animals are dosed on Day 9 andcollections are performed 12 hours later.Materials and MethodsMice

Normal male C57Bl/6 mice between the ages of 6-8 weeks old, weighing20-24 g, were obtained from Charles River Laboratories. The mice wererandomized into five groups of six mice each, and housed in groups of8-15 per cage, and acclimatized for at least three days prior toentering the study. Animal rooms were set to maintain a minimum of 12 to15 air changes per hour, with an automatic timer for a light/dark cycleof 12 hours on/off, and fed with Labdiet 5053 sterile rodent chow, withwater administered ad libitum.

Cecal Cannulation

The animals were placed under isoflurane anesthesia, with the cecumexposed via a midline incision in the abdomen. A small point incisionwas made in the distal cecum, where 1-2 cm of the cannula was inserted.The incision was closed with a purse string suture using 5-0 silk. Anincision was then made in the left abdominal wall through which thedistal end of the cannula was inserted and pushed subcutaneously to thedorsal aspect of the back. The site was then washed copiously withwarmed saline prior to closing the abdominal wall. A small incision wasalso made in the skin of the back between the shoulder blades, exposingthe tip of the cannula. The cannula was secured in place using suture,wound clips, and tissue glue. All animals were administered 1 mL of warmsterile saline (subcutaneous injection) and were monitored closely untilrecovery before returning to their cage. All animals were administered0.6 mg/kg BID buprenorphine for the first 3 days, and Baytril® at 10mg/Kg every day for the first 5 days post-surgery.

Disease Induction

Colitis was induced on Day 0 via addition of 3% DSS (MP Biomedicals, Cat#0260110) to the drinking water. Fresh DSS/water solutions was providedon Day 3 and any of the remaining original DSS solution is discarded.

Body Weight and Survival

Animals were observed daily (weight, morbidity, survival, presence ofdiarrhea and/or bloody stool) in order to assess possible differencesamong treatment groups and/or possible toxicity resulting from thetreatments.

Animals Found Dead or Moribund

Animals were monitored on a daily basis. Animals exhibiting weight lossgreater than 30% were euthanized, and samples were not collected fromthese animals.

Dosing

Animals were dosed with test-article via oral gavage (PO) or intracecalinjection (IC) once on Day 9 as indicated in Table 12. Animals in Group0 were not dosed. Animals in Groups 2 and 4 were dosed PO with SMAD7antisense. Animals in Groups 3 and 5 were dosed IC with SMAD7 antisense.

Sacrifice

All animals were euthanized by CO₂ inhalation 12 hours after dosing, onDay 10.

Sample Collection

Intestinal contents, peripheral blood and tissue were collected atsacrifice on Day 10, as follows:

Blood/Plasma

Terminal blood was collected into two K₂EDTA tubes and processed forplasma. The approximate volume of each blood sample was recorded priorto centrifugation. Both plasma and pellet samples were snap-frozen inliquid nitrogen and stored at −80° C. The first pellet sample (sample 1)was used for fluoremetry. The second pellet sample (sample 2) was usedfor RP-HPLC.

Cecum Contents

Cecum contents was removed and contents were split into two aliquots.Both aliquots were weighed and snap frozen in separate cryovials inliquid nitrogen. The first sample (sample 1) was used for fluorometry.The second sample (sample 2) was used for RP-HPLC.

Cecum

The cecum was excised and bisected longitudinally; each piece wasseparately weighed and snap-frozen. The first sample (sample 1) was usedfor fluoremetry. The second sample (sample 2) was used for RP-HPLC.

Colon Contents

Colon contents were removed and contents were split into two aliquots.Both aliquots were weighed and snap frozen in separate cryovials inliquid nitrogen. The first sample (sample 1) was used for fluorometry.The second sample (sample 2) was used for RP-HPLC.

Colon

The colon was rinsed, and the most proximal 2 cm of colon was collectedand bisected longitudinally. Each piece was separately weighed andflash-frozen in liquid nitrogen. The first sample (sample 1) was usedfor fluorometry. The second sample (sample 2) was used for RP-HPLC.

SMAD7 Antisense Bioanalysis

Samples flash-frozen for fluoremetry were homogenized in 0.5 mL bufferRLT+ (Qiagen). Homogenate was centrifuged (4000×g; 10 minutes), andsupernatant was collected. Forty microliters of the sample was diluted1:6 in 200 μL of bicarbonate solution and 100 μL of diluted supernatantwas analyzed on a fluorescent plate reader (485 excitation; 535emission) in duplicate.

Prior to the above, assay development was performed as follows. Samples(as indicated in Sample Collection) were harvested from a naïve animaland flash-frozen. Samples were then homogenized in 0.5 mL buffer RLT+,homogenate was centrifuged (4000×g; 10 minutes) and supernatant wascollected and diluted 1:6 with bicarbonate solution (i.e., 0.5 mLsupernatant was added to 2.5 mL of PBS). An aliquot (0.200 mL (90 μL foreach duplicate) of each diluted sample was pipetted into 15 (14 dilutionof FAM-AS-SAMD7+ blank control) Eppendorf tubes. One tube was set-asideto be used as a blank sample. Ten microliters of fluorescently-labeledSMAD7 antisense was then spiked into all other sample to achieve finalconcentrations of 50 μg/mL, 16.67 μg/mL, 5.56 μg/mL, 1.85 μg/mL, 0.62μg/mL, 0.21 μg/mL, 0.069 μg/mL, 0.023 μg/mL, 7.6 ng/mL, 2.5 ng/mL, 0.847ng/mL, 0.282 ng/mL, 0.094 ng/mL, and 0.024 ng/mL respectively. Thefluorescently-labeled SMAD7 antisense was prepared and serially dilutedsuch that the volume added to each organ homogenate sample was the samefor each of the above concentrations. These samples were analyzed on afluorescent plate reader (485 excitation; 535 emission) in duplicate.

Processing for RP-HPLC

Samples flash-frozen for RP-HPLC were homogenized in bufferRLT+(Qiagen). Homogenate was centrifuged (4000×g; 10 minutes), andsupernatant was used to perform RP-HPLC analysis.

Results

The data in FIGS. 73 and 74 show that significantly more SMAD7 antisenseoligonucleotide was present in cecum tissue and colon tissue for micewith or without DSS treatment that were intra-cecally administered theSMAD7 antisense oligonucleotide as compared to mice with or without DSStreatment that were orally administered the SMAD7 antisenseoligonucleotide. The data in FIG. 75 show that there is about the samelevel of SMAD7 antisense oligonucleotide in the cecum contents of micewith or without DSS treatment that were orally or intra-cecallyadministered the SMAD7 antisense oligonucleotide. No SMAD7 antisenseoligonucleotide was found in the plasma or white blood cell pellet ofSMAD7 antisense oligonucleotide treated mice.

Example 10. Comparison of the Tissue, Plasma, and GI ContentPharmacokinetics of Tacrolimus Through Oral Vs. Intra-Cecal IngestibleDevice Delivery in Yorkshire-Cross Farm Swine

The primary objective of this study was to compare the tissue, plasma,rectal sample, and GI content pharmacokinetics of tacrolimus throughoral versus intra-cecal ingestible device delivery in normalYorkshire-Cross farm swine.

This study compares the effects of administration of: a singleintra-cecal administration of an ingestible device containing 0.8 mLsterile vehicle solution (80% alcohol, 20% castor oil (HCO-60)); asingle oral dose of tacrolimus at 4 mg/0.8 mL (in sterile vehiclesolution); and a single intra-cecal administration of an ingestibledevice containing either 1 mg/0.8 mL (in sterile vehicle solution), 2mg/0.8 mL (in sterile vehicle solution), or 4 mg/0.8 mL (in sterilevehicle solution).

This study employed five groups of three female swine weighingapproximately 45 to 50 kg at study start. Swine were randomly placedinto animal rooms/pens as they are transferred from the delivery vehiclewithout regard to group. Group numbers were assigned to the rooms inorder of room number. No further randomization procedure was employed.The study design is provided in Table 13.

TABLE 13 Study Design Table Days Pre-Dose Hours Post-dose Group sizeDose Route −11 −10 −5 −1 1 0.5 1 2 3 4 6 12 General Fast • • Food/Waterad libidum • • • • • • • • • • Observations clinical observations Day−10~5 & • • • • • • • • • • body weight* Day 1 • • • • Treatments(Groups) 1. Vehicle control n = 3 0.8 mL (20% IC HCO-60, 80% EtOH)Surgical placement of • IC port** Euthanized (1 Ingestible n = 3 Device)2. Tacrolimus (PO) n = 3 4 mg in 0.8 mL Oral • Surgical placement of0.08 mg/kg • IC port** Euthanized (solution) n = 3 3. Tacrolimus (IC) n= 3 1 mg in 0.8 mL IC • Surgical placement of 0.02 mg/kg • IC port**Euthanized (1 Ingestible n = 3 Device) 4. Tacrolimus (IC) n = 3 2 mg in0.8 mL IC • Surgical placement of 0.04 mg/kg • IC port** Euthanized (1Ingestible n = 3 Device) 5. Tacrolimus (IC) n = 3 4 mg in 0.8 mL IC •Surgical placement of 0.08 mg/kg • IC port** Euthanized (1 Ingestible n= 3 Device) Tacrolimus (required) 20 mg Samples***** Plasma cephalic, •• • • • • • Rectal contents jugular or • • • • Tissue*** x5 catheter •Luminal contents**** x5 rectal • necropsy necropsy Analysis (AgriluxTotal Charles River) Samples Plasma [Tacrolimus] 105 15 15 15 15 15 1515 Rectal contents [Tacrolimus]  60 15 15 15 15 Tissue (intact)***[Tacrolimus] 105 105 Luminal contents [Tacrolimus]  75 75 Tissue afterremoving luminal content [Tacrolimus]  75 75

Animals in Group 1 received an ingestible device containing 0.8 mL ofvehicle solution (80% alcohol, 20% HCO-60). Animals in Group 2 receivedorally 4 mL liquid formulation of tacrolimus at 4 mg/0.8 mL per animal(Prograf: 5 mg/mL). Animals in Group 3 received intra-cecally aningestible device containing tacrolimus at 1 mg in 0.8 mL per ingestibledevice. Animals in Group 4 received intra-cecally an ingestible devicecontaining tacrolimus at 2 mg in 0.8 mL per ingestible device. Animalsin Group 5 received intra-cecally an ingestible device containingtacrolimus at 4 mg in 0.8 mL per ingestible device. To control forpotential confounding effects of the surgery, all groups fast on Day −11at least 24 hr before being subjected to anesthesia followed by surgicalplacements of a cecal port by a veterinary surgeon at Day −10. Allanimals were fasted for at least 12 hr prior to dosing on Day 1. Animalswere dosed via either intra-cecal dosing (IC) or oral dosing (PO) at Day1 (between 6-8 p.m.). All animals resumed feeding at approximately 4hours after dose (11-12 p.m. after dosing).

Animals in Group 1 (Vehicle Control) were administered a singleintra-cecal ingestible device containing 0.8 mL Vehicle solution (80%alcohol, 20% castor oil (HCO-60) on Day 1. On Day −10 the animals wereanesthetized, and a veterinary surgeon surgically placed an intra-cecalport in each animal. On Day 1, each animal was placed into a sling thena single intra-cecal ingestible device containing 0.8 mL vehiclesolution (80% alcohol, 20% castor oil (HCO-60)) is introduced by theveterinary surgeon into the cecum via the cecal port in each animal.Following ingestible device placement, the animals were removed from theslings and placed back into their pens with water. All animals resumedfeeding at approximately 4 hours after dose. Samples of rectal contentswere collected for pharmacokinetic analyses from each animal at each of1, 3, 6, and 12 hours post-ingestible device placement using a fecalswab (rectal swab). A total of 60 samples were collected.

Approximately 200˜400 mg of rectal content were collected, if available,with a fecal swab (Copan Diagnostics Nylon Flocked Dry Swabs, 502CS01).The fecal swab was pre-weighed and weighed after collection in thecollection tube (Sterile Tube and Cap No Media, PFPM913S), and thesample weight was recorded. The fecal swab was broken via thebreakpoint, and was stored in the collection tube, and immediatelyfrozen at −70° C. Whole blood (2 mL) was collected into K₂EDTA coatedtubes for pharmacokinetics at each time-point of pre-dose and 1, 2, 3,4, 6 and 12 hours post-dose. Immediately following euthanasia, tissuewas collected. A total of 105 samples were collected.

For tissue necropsy, small intestine fluid and cecal fluid werecollected separately from all the animals into two separate squareplastic bottles, and stored at −20° C. The length and diameter of thececum and the colon was measured from one animal in each group andrecorded for reference. Tissues were collected for pharmacokineticanalyses and include mesenteric lymph nodes, a Peyer's Patch, and fivegastrointestinal sections, including cecum, proximal colon, transversecolon, distal colon, and rectum. All samples were weighed, and thetissue sample weights were recorded. In each of the fivegastrointestinal sections, tissue samples were collected in threedifferent areas where the mucosal surface was visible and not covered byluminal content by using an 8.0-mm punch biopsy tool. Around 3 grams ofthe total punched sample were collected into a pre-weighed 15-mL conicaltube, and the tissue weight was recorded. Three mesenteric lymph nodeswere collected from different areas and weighed. At least one Peyer'sPatch was collected and weighed. Tissues were snap-frozen in liquidnitrogen and stored frozen at approximately −70° C. or below (total of105 samples).

Luminal contents were collected for pharmacokinetic analyses from thesurface of the tissue from each of five gastrointestinal sections:cecum, proximal colon, transverse colon, distal colon, and rectum (totalof 75). The contents were collected in pre-weighed 15-mL conical tubesand the sample weights were recorded. Samples were snap-frozen in liquidnitrogen stored frozen at approximately −70° C. or below.

After removing the luminal content, another set of tissue samples from 3different areas were collected via an 8.0-mm punch biopsy in eachsection of the five tissue gastrointestinal sections described above.Around 3 grams of the total punched sample were collected into apre-weighed 15-mL conical tube, and the tissue weight was recorded(total of 75). Tissues were snap-frozen in liquid nitrogen and storedfrozen at approximately −70° C. or below.

A 30-cm length of jejunum (separated into two 15 cm lengths), and theremaining distal and transverse colon tissue sample (after tissue andluminal content were collected for PK) were collected in one animal ineach group of treatment, snap-frozen in liquid nitrogen and storedfrozen at approximately −70° C. or below. All samples forpharmacokinetic analyses were stored on dry ice before analyses.

Group 2 animals were administered a single oral dose of tacrolimus at 1mg/0.8 mL (in the vehicle solution) on Day 1. Plasma, rectal contentsample, tissue collection, GI content collection and relatedprocedures/storage/shipments was the same as those employed in Group 1.

Group 3 animals were administered a single intra-cecal ingestible devicecontaining tacrolimus at 0.5 mg/0.8 mL (in the vehicle solution) on Day1 by a veterinary surgeon. Plasma, rectal content sample, tissuecollection, GI content collection and relatedprocedures/storage/shipments was the same as those employed in Group 1.All samples were analyzed for tacrolimus.

Group 4 animals were administered a single intra-cecal ingestible deviceof tacrolimus at 2 mg/0.8 mL (in sterile vehicle solution) on Day 1 by aveterinary surgeon. Plasma, rectal content sample, tissue collection, GIcontent collection and related procedures/storage/shipments were thesame as those employed in Group 1. All samples were analyzed fortacrolimus.

Group 5 animals are administered a single intra-cecal ingestible devicecontaining tacrolimus at 4 mg/0.8 mL (in the vehicle solution) on Day 1by a veterinary surgeon. Plasma, rectal content sample, tissuecollection, GI content collection and relatedprocedures/storage/shipments were the same as those employed in Group 1.All samples were analyzed for tacrolimus.

Detailed clinical observations were conducted daily from Day −10 to −5,and on Day 1. Additional pen-side observations were conducted at leastonce each day. The animals remained under constant clinical observationfor the entire 12 hours from dose until euthanasia. Body weights werecollected on Day −10, Day −5, and pre-dose on Day 1. Animals wereeuthanized via injection of a veterinarian-approved euthanasia.

Test Article and Formulation

1. Vehicle Solution, 20 mL

Description: 80% alcohol, 20% PEG-60 castor oil

Physical characteristics: clear liquid solution.

2. Prograf (Tacrolimus Injection), 10 Ampules

Description: A sterile solution containing the equivalent of 5 mganhydrous tacrolimus in 1 mL. Tacrolimus is macrolide immunosuppressantand the active ingredient of Prograf. 0.8 mL of Prograf (5 mg/mL) wasadministrated through oral gavage per animal in group 2. Prograf (5mg/mL) was diluted 2× folds (2.5 mg/mL) and 4× folds (1.25 mg/mL) byusing vehicle solution. 0.8 mL of each concentration, 1.25 mg/mL, 2.5mg/mL, and 5 mg/mL of Prograf, was injected into a DSS ingestible devicefor group 3, 4, and 5.Formulation: Each mL contained polyoxyl 60 hydrogenated castor oil(HCO-60), 200 mg, and dehydrated alcohol, USP, 80.0% v/v.Physical characteristics: clear liquid solution.3. DDS Ingestible Device Containing TacrolimusDescription: Three (3) DDS ingestible devices containing vehiclesolution for Group 1, three (3) DSS ingestible devices containing 1 mgtacrolimus for Group 3, three (3) DDS ingestible devices containing 2 mgtacrolimus for Group 4, and three (3) DDS ingestible devices containing4 mg tacrolimus for Group 5.Acclimation

Animals were acclimated prior to study initiation for at least 7 days.Animals in obvious poor health were not placed on study.

Concurrent Medication

Other than veterinary-approved anesthetics and medications used duringsurgery to install the ileocecal ports, or for vehicle or test articleadministration, and analgesia and antibiotics post-surgery, no furthermedications were employed.

Feed

All swine were fasted at least 24 hours before being anesthetized andproperly medicated for surgery or overnight before dosing. Otherwise,animals were fed ad-libitum. Tap water was pressure-reduced and passedthrough a particulate filter, then a carbon filter prior to supply to anautomatic watering system. Water was supplied ad libitum. There were noknown contaminants in the feed or water that would be expected tointerfere with this study.

Results

The data in FIG. 76 show that the mean concentration of tacrolimus inthe cecum tissue and the proximate colon tissue were higher in swinethat were inta-cecally administered tacrolimus as compared to swine thatwere orally administered tacrolimus. These data suggest that intra-cecaladministration of tacrolimus is able to locally deliver tacrolimus tothe tissues in the GI tract of a mammal, while not decreasing thesystemic immune system of a mammal.

Example 11. Comparison of the Tissue, Plasma, and GI ContentPharmacokinetics of Adalimumab Through SC Vs. Intra-Cecal IngestibleDevice Delivery in Yorkshire-Cross Farm Swine in DSS-Induced Colitis

The purpose of this non-Good Laboratory Practice (GLP) study is toexplore the PK/PD and bioavailability of adalimumab when applied toDSS-induced colitis in Yorkshire-cross farm swine. All animals arerandomized into groups of three. Animals are dosed once with adalimumabvia subcutaneous (SC), perirectal (PR), or intracecal (IC)administration.

The concentration of adalimumab and TNFα is measured in plasma at 1, 2,3, 4, 6, and 12 hours post-dose. The concentration of adalimumab ismeasured in rectal contents at 1, 3, 6, and 12 hours post-dose and inluminal content at 12 hours post-dose. Concentration of adalimumab andTNFα, HER2, and total protein is measured in gastrointestinal tissue,e.g., cecum sample (CAC), proximal colon sample (PCN), transverse colonsample (TCN), distal colon sample (DCNi) inflamed, distal colonnon-inflamed sample (DCNn), and rectum sample (RTM), at 12 hourspost-dose.

Example 12. Human Clinical Trial of Treatment of Ulcerative ColitisUsing Adalimumab

As a proof of concept, the patient population of this study is patientsthat (1) have moderate to severe ulcerative colitis, regardless ofextent, and (2) have had an insufficient response to a previoustreatment, e.g., a conventional therapy (e.g., 5-ASA, corticosteroid,and/or immunosuppressant) or a FDA-approved treatment. In thisplacebo-controlled eight-week study, patients are randomized. Allpatient undergo a colonoscopy at the start of the study (baseline) andat week 8. Patients enrolled in the study are assessed for clinicalstatus of disease by stool frequency, rectal bleeding, abdominal pain,physician's global assessment, and biomarker levels such as fecalcalprotectin and hsCRP. The primary endpoint is a shift in endoscopyscores from Baseline to Week 8. Secondary and exploratory endpointsinclude safety and tolerability, change in rectal bleeding score, changein abdominal pain score, change in stool frequency, change in partialMayo score, change in Mayo score, proportion of subjects achievingendoscopy remission, proportion of subjects achieving clinicalremission, change in histology score, change in biomarkers of diseasesuch as fecal calprotectin and hsCRP, level of adalimumab in theblood/tissue/stool, change in cytokine levels (e.g., TNFα, IL-6) in theblood and tissue.

FIG. 72 describes an exemplary process of what would occur in clinicalpractice, and when, where, and how the ingestible device will be used.Briefly, a patient displays symptoms of ulcerative colitis, includingbut not limited to: diarrhea, bloody stool, abdominal pain, highc-reactive protein (CRP), and/or high fecal calprotectin. A patient mayor may not have undergone a colonoscopy with diagnosis of ulcerativecolitis at this time. The patient's primary care physician refers thepatient. The patient undergoes a colonoscopy with a biopsy, CT scan,and/or MRI. Based on this testing, the patient is diagnosed withulcerative colitis. Most patients are diagnosed with ulcerative colitisby colonoscopy with biopsy. The severity based on clinical symptoms andendoscopic appearance, and the extent, based on the area of involvementon colonoscopy with or without CT/MRI is documented. Treatment isdetermined based on diagnosis, severity and extent.

For example, treatment for a patient that is diagnosed with ulcerativecolitis is an ingestible device programmed to release a single bolus ofa therapeutic agent, e.g., 40 mg adalimumab, in the cecum or proximal tothe cecum. Prior to administration of the treatment, the patient isfasted overnight and is allowed to drink clear fluids. Four hours afterswallowing the ingestible device, the patient can resume a normal diet.An ingestible device is swallowed at the same time each day. Theingestible device is not recovered.

In some embodiments, there may be two different ingestible devices: oneincluding an induction dose (first 8 to 12 weeks) and a differentingestible device including a different dose or a different dosinginterval.

In some examples, the ingestible device can include a mapping tool,which can be used after 8 to 12 weeks of induction therapy, to assessthe response status (e.g., based on one or more of the following: druglevel, drug antibody level, biomarker level, and mucosal healingstatus). Depending on the response status determined by the mappingtool, a subject may continue to receive an induction regimen ormaintenance regimen of adalimumab.

In different clinical studies, the patients may be diagnosed withCrohn's disease and the ingestible devices (including adalimumab) can beprogrammed to release adalimumab in the cecum, or in both the cecum andtransverse colon.

In different clinical studies, the patients may be diagnosed withilleocolonic Crohn's disease and the ingestible devices (includingadalimumab) can be programmed to release adalimumab in the late jejunumor in the jejunum and transverse colon.

Example 13

An ingestible medical device according to the disclosure (“TLC1”) wastested on 20 subjects to investigate its localization ability. TLC1 wasa biocompatible polycarbonate ingestible device that contained a powersupply, electronics and software. An onboard software algorithm usedtime, temperature and reflected light spectral data to determine thelocation of the ingestible device as it traveled the GI tract. Theingestible device is 0.51×1.22 inches which is larger than a vitaminpill which is 0.4×0.85 inches. The subjects fasted overnight beforeparticipating in the study. Computerized tomography (“CT”) were used asa basis for determining the accuracy of the localization data collectedwith TLC1. One of the 20 subjects did not follow the fasting rule. CTdata was lacking for another one of the 20 subjects. Thus, these twosubjects were excluded from further analysis. TLC1 sampled RGB data(radially transmitted) every 15 seconds for the first 14 hours after itentered the subject's stomach, and then samples every five minutes afterthat until battery dies. TLC1 did not start to record optical data untilit reached the subject's stomach. Thus, there was no RGB-based data forthe mouth-esophagus transition for any of the subjects.

In addition, a PillCam® SB (Given Imaging) device was tested on 57subjects. The subjects fasted overnight before joining the study.PillCam videos were recorded within each subject. The sampling frequencyof PillCam is velocity dependent. The faster PillCam travels, the fasterit would sample data. Each video is about seven to eight hours long,starting from when the ingestible device was administrated into thesubject's mouth. RGB optical data were recorded in a table. A physicianprovided notes on where stomach-duodenum transition and ileum-cecumtransition occurred in each video. Computerized tomography (“CT”) wasused as a basis for determining the accuracy of the localization datacollected with PillCam.

Esophagus-Stomach Transition

For TLC1, it was assumed that this transition occurred one minute afterthe patient ingested the device. For PillCam, the algorithm was asfollows:

-   -   1. Start mouth-esophagus transition detection after ingestible        device is activated/administrated    -   2. Check whether Green <102.3 and Blue <94.6        -   a. If yes, mark as mouth-esophagus transition        -   b. If no, continue to scan the data    -   3. After detecting mouth-esophagus transition, continue to        monitor Green and Blue signals for another 30 seconds, in case        of location reversal        -   a. If either Green >110.1 or Blue >105.5, mark it as            mouth-esophagus location reversal        -   b. Reset the mouth-esophagus flag and loop through step 2            and 3 until the confirmed mouth-esophagus transition            detected    -   4. Add one minute to the confirmed mouth-esophagus transition        and mark it as esophagus-stomach transition

For one of the PillCam subjects, there was not a clear cut differencebetween the esophagus and stomach, so this subject was excluded fromfuture analysis of stomach localization. Among the 56 valid subjects, 54of them have correct esophagus-stomach transition localization. Thetotal agreement is 54/56=96%. Each of the two failed cases had prolongedesophageal of greater than one minute. Thus, adding one minute tomouth-esophagus transition was not enough to cover the transition inesophagus for these two subjects.

Stomach-Duodenum

For both TLC1 and PillCam, a sliding window analysis was used. Thealgorithm used a dumbbell shape two-sliding-window approach with atwo-minute gap between the front (first) and back (second) windows. Thetwo-minute gap was designed, at least in part, to skip the rapidtransition from stomach to small intestine and capture the smallintestine signal after ingestible device settles down in smallintestine. The algorithm was as follows:

-   -   1. Start to check for stomach-duodenum transition after        ingestible device enters stomach    -   2. Setup the two windows (front and back)        -   a. Time length of each window: 3 minutes for TLC1; 30            seconds for PillCam        -   b. Time gap between two windows: 2 minutes for both devices        -   c. Window sliding step size: 0.5 minute for both devices    -   3. Compare signals in the two sliding windows        -   a. If difference in mean is higher than 3 times the standard            deviation of Green/Blue signal in the back window            -   i. If this is the first time ever, record the mean and                standard deviation of signals in the back window as                stomach reference            -   ii. If mean signal in the front window is higher than                stomach reference signal by a certain threshold (0.3 for                TLC1 and 0.18 for PillCam), mark this as a possible                stomach-duodenum transition        -   b. If a possible pyloric transition is detected, continue to            scan for another 10 minutes in case of false positive flag            -   i. If within this 10 minutes, location reversal is                detected, the previous pyloric transition flag is a                false positive flag. Clear the flag and continue to                check            -   ii. If no location reversal has been identified within                10 minutes following the possible pyloric transition                flag, mark it as a confirmed pyloric transition        -   c. Continue monitoring Green/Blue data for another 2 hours            after the confirmed pyloric transition, in case of location            reversal            -   i. If a location reversal is identified, flag the                timestamp when reversal happened and then repeat steps                a-c to look for the next pyloric transition            -   ii. If the ingestible device has not gone back to                stomach 2 hours after previously confirmed pyloric                transition, stops location reversal monitoring and                assume the ingestible device would stay in intestinal                area

For TLC1, one of the 18 subjects had too few samples (<3 minutes) takenin the stomach due to the delayed esophagus-stomach transitionidentification by previously developed localization algorithm. Thus,this subject was excluded from the stomach-duodenum transition algorithmtest. For the rest of the TLC1 subjects, CT images confirmed that thedetected pyloric transitions for all the subjects were located somewherebetween stomach and jejunum. Two out of the 17 subjects showed that theingestible device went back to stomach after first the firststomach-duodenum transition. The total agreement between the TLC1algorithm detection and CT scans was 17/17=100%.

For one of the PillCam subjects, the ingestible device stayed in thesubject's stomach all the time before the video ended. For another twoof the PillCam subjects, too few samples were taken in the stomach torun the localization algorithm. These three PillCam subjects wereexcluded from the stomach-duodenum transition localization algorithmperformance test. The performance summary of pyloric transitionlocalization algorithm for PillCam was as follows:

-   -   1. Good cases (48 subjects):        -   a. For 25 subjects, our detection matches exactly with the            physician's notes        -   b. For 19 subjects, the difference between the two            detections is less than five minutes        -   c. For four subjects, the difference between the two            detections is less than 10 minutes (The full transition            could take up to 10 minutes before the G/B signal settled)    -   2. Failed cases (6 subjects):        -   a. Four subjects had high standard deviation of Green/Blue            signal in the stomach        -   b. One subject had bile in the stomach, which greatly            affected Green/Blue in stomach        -   c. One subject had no Green/Blue change at pyloric            transition

The total agreement for the PillCam stomach-duodenum transitionlocalization algorithm detection and physician's notes was 48/54=89%.

Duodenum-Jejenum Transition

For TLC1, it was assumed that the device left the duodenum and enteredthe jejenum three minutes after it was determined that the deviceentered the duodenum. Of the 17 subjects noted above with respect to theTLC1 investigation of the stomach-duodenum transition, 16 of thesubjects mentioned had CT images that confirmed that theduodenum-jejenum transition was located somewhere between stomach andjejunum. One of the 17 subjects had a prolonged transit time induodenum. The total agreement between algorithm detection and CT scanswas 16/17=94%.

For PillCam, the duodenum-jejenum transition was not determined.

Jejenum-Ileum Transition

It is to be noted that the jejunum is redder and more vascular thanileum, and that the jejenum has a thicker intestine wall with moremesentery fat. These differences can cause various optical responsesbetween jejunum and ileum, particularly for the reflected red lightsignal. For both TLC1 and PillCam, two different approaches wereexplored to track the change of red signal at the jejunum-ileumtransition. The first approach was a single-sliding-window analysis,where the window is 10 minutes long, and the mean signal was comparedwith a threshold value while the window was moving along. The secondapproach was a two-sliding-window analysis, where each window was 10minutes long with a 20 minute spacing between the two windows. Thealgorithm for the jejunum-ileum transition localization was as follows:

-   -   1. Obtain 20 minutes of Red signal after the duodenum jejenum        transition, average the data and record it as the jejunum        reference signal    -   2. Start to check the jejunum-ileum transition 20 minutes after        the device enters the jejunum        -   a. Normalize the newly received data by the jejunum            reference signal        -   b. Two approaches:            -   i. Single-sliding-window analysis                -   Set the transition flag if the mean of reflected red                    signal is less than 0.8            -   ii. Two-sliding-window analysis:                -   Set the transition flag if the mean difference in                    reflected red is higher than 2× the standard                    deviation of the reflected red signal in the front                    window

For TLC1, 16 of the 18 subjects had CT images that confirmed that thedetected jejunum-ileum transition fell between jejunum and cecum. Thetotal agreement between algorithm and CT scans was 16/18=89%. This wastrue for both the single-sliding-window and double-sliding-windowapproaches, and the same two subjects failed in both approaches.

The performance summary of the jejunum-ileum transition detection forPillCam is listed below:

-   -   1. Single-sliding-window analysis:        -   a. 11 cases having jejunum-ileum transition detected            somewhere between jejunum and cecum        -   b. 24 cases having jejunum-ileum transition detected after            cecum        -   c. 19 cases having no jejunum-ileum transition detected        -   d. Total agreement: 11/54=20%    -   2. Two-sliding-window analysis:        -   a. 30 cases having jejunum-ileum transition detected            somewhere between jejunum and cecum        -   b. 24 cases having jejunum-ileum transition detected after            cecum        -   c. Total agreement: 30/54=56%            Ileum-Cecum Transition

Data demonstrated that, for TLC1, mean signal of reflected red/greenprovided the most statistical difference before and after theileum-cecum transition. Data also demonstrated that, for TLC1, thecoefficient of variation of reflected green/blue provided the moststatistical contrast at ileum-cecum transition. The analysis based onPillCam videos showed very similar statistical trends to those resultsobtained with TLC1 device. Thus, the algorithm utilized changes in meanvalue of reflected red/green and the coefficient of variation ofreflected green/blue. The algorithm was as follows:

-   -   1. Start to monitor ileum-cecum transition after the ingestible        device enters the stomach    -   2. Setup the two windows (front (first) and back (second))        -   a. Use a five-minute time length for each window        -   b. Use a 10-minute gap between the two windows        -   c. Use a one-minute window sliding step size    -   3. Compare signals in the two sliding windows        -   a. Set ileum-cecum transition flag if            -   i. Reflected red/green has a significant change or is                lower than a threshold            -   ii. Coefficient of variation of reflected green/blue is                lower than a threshold        -   b. If this is the first ileum-cecum transition detected,            record average reflected red/green signal in small intestine            as small intestine reference signal        -   c. Mark location reversal (i.e. ingestible device returns to            terminal ileum) if            -   i. Reflected red/green is statistically comparable with                small intestine reference signal            -   ii. Coefficient of variation of reflected green/blue is                higher than a threshold        -   d. If a possible ileum-cecum transition is detected,            continue to scan for another 10 minutes for TLC1 (15 minutes            for PillCam) in case of false positive flag            -   i. If within this time frame (10 minutes for TLC1, 15                minutes for PillCam), location reversal is detected, the                previous ileum-cecum transition flag is a false positive                flag. Clear the flag and continue to check            -   ii. If no location reversal has been identified within                this time frame (10 minutes for TLC1, 15 minutes for                PillCam) following the possible ileum-cecum transition                flag, mark it as a confirmed ileum-cecum transition        -   e. Continue monitoring data for another 2 hours after the            confirmed ileum-cecum transition, in case of location            reversal            -   i. If a location reversal is identified, flag the                timestamp when reversal happened and then repeat steps                a-d to look for the next ileum-cecum transition            -   ii. If the ingestible device has not gone back to small                intestine 2 hours after previously confirmed ileum-cecum                transition, stop location reversal monitoring and assume                the ingestible device would stay in large intestinal                area

The flag setting and location reversal criteria particularly designedfor TLC1 device were as follows:

-   -   1. Set ileum-cecum transition flag if        -   a. The average reflected red/Green in the front window is            less than 0.7 or mean difference between the two windows is            higher than 0.6        -   b. And the coefficient of variation of reflected green/blue            is less than 0.02    -   2. Define as location reversal if        -   a. The average reflected red/green in the front window is            higher than small intestine reference signal        -   b. And the coefficient of variation of reflected green/blue            is higher than 0.086

For TLC1, 16 of the 18 subjects had CT images that confirmed that thedetected ileum-cecum transition fell between terminal ileum and colon.The total agreement between algorithm and CT scans was 16/18=89%.Regarding those two subject where the ileum-cecum transitionlocalization algorithm failed, for one subject the ileum-cecumtransition was detected while TLC1 was still in the subject's terminalileum, and for the other subject the ileum-cecum transition was detectedwhen the device was in the colon.

Among the 57 available PillCam endoscopy videos, for three subjects theendoscopy video ended before PillCam reached cecum, and another twosubjects had only very limited video data (less than five minutes) inthe large intestine. These five subjects were excluded from ileum-cecumtransition localization algorithm performance test. The performancesummary of ileum-cecum transition detection for PillCam is listed below:

-   -   1. Good cases (39 subjects):        -   a. For 31 subjects, the difference between the PillCam            detection and the physician's notes was less than five            minutes        -   b. For 3 subjects, the difference between the PillCam            detection and the physician's notes was less than 10 minutes    -   c. For 5 subjects, the difference between the PillCam detection        and the physician's notes was less than 20 minutes (the full        transition can take up to 20 minutes before the signal settles)    -   2. Marginal/bad cases (13 subjects):        -   a. Marginal cases (9 subjects)            -   i. The PillCam ileum-cecum transition detection appeared                in the terminal ileum or colon, but the difference                between the two detections was within one hour        -   b. Failed cases (4 subjects)            -   i. Reasons of failure:                -   1. The signal already stabilized in the terminal                    ileum                -   2. The signal was highly variable from the entrance                    to exit                -   3. There was no statistically significant change in                    reflected red/green at ileum-cecum transition

The total agreement between ileocecal transition localization algorithmdetection and the physician's notes is 39/52=75% if considering goodcases only. Total agreement including possibly acceptable cases is48/52=92.3%

Cecum-Colon Transition

Data demonstrated that, for TLC1, mean signal of reflected red/greenprovided the most statistical difference before and after thececum-colon transition. Data also demonstrated that, for TLC1, thecoefficient of variation of reflected blue provided the most statisticalcontrast at cecum-colon transition. The same signals were used forPillCam. The cecum-colon transition localization algorithm was asfollows:

-   -   1. Obtain 10 minutes of reflected red/green and reflected blue        signals after ileum-cecum transition, average the data and        record it as the cecum reference signals    -   2. Start to check cecum-colon transition after ingestible device        enters cecum (The cecum-colon transition algorithm is dependent        on the ileum-cecum transition flag)        -   a. Normalize the newly received data by the cecum reference            signals        -   b. Two-sliding-window analysis:            -   i. Use two adjacent 10 minute windows            -   ii. Set the transition flag if any of the following                criteria were met                -   The mean difference in reflected red/green was more                    than 4× the standard deviation of reflected                    red/green in the back (second) window                -   The mean of reflected red/green in the front (first)                    window was higher than 1.03                -   The coefficient of variation of reflected blue                    signal in the front (first) window was greater than                    0.23

The threshold values above were chosen based on a statistical analysisof data taken by TLC1.

For TLC1, 15 of the 18 subjects had the cecum-colon transition detectedsomewhere between cecum and colon. One of the subjects had thececum-colon transition detected while TLC1 was still in cecum. The othertwo subjects had both wrong ileum-cecum transition detection and wrongcecum-colon transition detection. The total agreement between algorithmand CT scans was 15/18=83%.

For PillCam, for three subjects the endoscopy video ended before PillCamreached cecum, and for another two subjects there was very limited videodata (less than five minutes) in the large intestine. These fivesubjects were excluded from cecum-colon transition localizationalgorithm performance test. The performance summary of cecum-colontransition detection for PillCam is listed below:

-   -   1. 27 cases had the cecum-colon transition detected somewhere        between the cecum and the colon    -   2. one case had the cecum-colon transition detected in the ileum    -   3. 24 cases had no cecum-colon transition localized        The total agreement: 27/52=52%.        The following table summarizes the localization accuracy        results.

Transition TLC1 PillCam Stomach-Duodenum 100% (17/17) 89% (48/54)Duodenum-Jejenum  94% (16/17) N/A Ileum-Cecum  89% (16/18) 75% (39/52)Ileum-terminal 100% (18/18) 92% (48/52) ileum/cecum/colon

Exemplary Embodiments

The following exemplary embodiments 1)-94) are provided herein:

1) A method of treating a disease of the gastro-intestinal tract in asubject, comprising:

delivering a TLR agonist at a location in the gastrointestinal tract ofthe subject,

wherein the method comprises administering orally to the subject apharmaceutical composition comprising a therapeutically effective amountof the TLR agonist.

2) The method of exemplary embodiment 1, wherein the disease of the GItract is an inflammatory bowel disease.

3) The method of exemplary embodiment 1, wherein the disease of the GItract is ulcerative colitis.

4) The method of exemplary embodiment 1, wherein the disease of the GItract is Crohn's disease.

5) The method of any one of exemplary embodiments 1, 2, or 3, 4, whereinthe TLR agonist is delivered at a location in the large intestine of thesubject.

6) The method of exemplary embodiment 5, wherein the location is in theproximal portion of the large intestine.

7) The method of exemplary embodiment 5, wherein the location is in thedistal portion of the large intestine.

8) The method of any one of exemplary embodiments 1, 2, or 3, 4, whereinthe TLR agonist is delivered at a location in the ascending colon of thesubject.

9) The method of exemplary embodiment 8, wherein the location is in theproximal portion of the ascending colon.

10) The method of exemplary embodiment 8, wherein the location is in thedistal portion of the ascending colon.

11) The method of any one of exemplary embodiments 1, 2, or 3, 4,wherein the TLR agonist is delivered at a location in the cecum of thesubject.

12) The method of exemplary embodiment 11, wherein the location is inthe proximal portion of the cecum.

13) The method of exemplary embodiment 11, wherein the location is inthe distal portion of the cecum.

14) The method of any one of exemplary embodiments 1, 2, or 3, 4,wherein the TLR agonist is delivered at a location in the sigmoid colonof the subject.

15) The method of exemplary embodiment 14, wherein the location is inthe proximal portion of the sigmoid colon.

16) The method of exemplary embodiment 14, wherein the location is inthe distal portion of the sigmoid colon.

17) The method of any one of exemplary embodiments 1, 2, or 3, 4,wherein the TLR agonist is delivered at a location in the transversecolon of the subject.

18) The method of exemplary embodiment 17, wherein the location is inthe proximal portion of the transverse colon.

19) The method of exemplary embodiment 17, wherein the location is inthe distal portion of the transverse colon.

20) The method of any one of exemplary embodiments 1, 2, or 3, 4,wherein the TLR agonist is delivered at a location in the descendingcolon of the subject.

21) The method of exemplary embodiment 20, wherein the location is inthe proximal portion of the descending colon.

22) The method of exemplary embodiment 20, wherein the location is inthe distal portion of the descending colon.

23) The method of any one of exemplary embodiments 1, 2, or 3, 4,wherein the TLR agonist is delivered at a location in the smallintestine of the subject.

24) The method of exemplary embodiment 23, wherein the location is inthe proximal portion of the small intestine.

25) The method of exemplary embodiment 23, wherein the location is inthe distal portion of the small intestine.

26) The method of any one of exemplary embodiments 1, 2, or 3, 4,wherein the TLR agonist is delivered at a location in the duodenum ofthe subject.

27) The method of exemplary embodiment 26, wherein the location is inthe proximal portion of the duodenum.

28) The method of exemplary embodiment 26, wherein the location is inthe distal portion of the duodenum.

29) The method of any one of exemplary embodiments 1, 2, or 3, 4,wherein the TLR agonist is delivered at a location in the jejunum of thesubject.

30) The method of exemplary embodiment 29, wherein the location is inthe proximal portion of the jejunum.

31) The method of exemplary embodiment 29, wherein the location is inthe distal portion of the jejunum.

32) The method of any one of exemplary embodiments 1, 2, or 3, 4,wherein the TLR agonist is delivered at a location in the ileum of thesubject.

33) The method of exemplary embodiment 32, wherein the location is inthe proximal portion of the ileum.

34) The method of exemplary embodiment 32, wherein the location is inthe distal portion of the ileum.

35) The method of any one of the preceding exemplary embodiments,wherein the location is proximate to one or more sites of disease.

36) The method of exemplary embodiment 35, further comprisingidentifying the one or more sites of disease by a method comprisingimaging of the gastrointestinal tract.

37) The method of any one of the preceding exemplary embodiments,wherein the TLR agonist is delivered to the location by mucosal contact.

38) The method of any one of the preceding exemplary embodiments,wherein the TLR agonist is delivered to the location by a process thatdoes not comprise systemic transport of the TLR agonist.

39) The method of any one of the preceding exemplary embodiments,wherein the amount of the TLR agonist that is administered is from about1 mg to about 300 mg.

40) The method of exemplary embodiment 39, wherein the amount of the TLRagonist that is administered is from about 1 mg to about 100 mg.

41) The method of exemplary embodiment 40, wherein the amount of the TLRagonist that is administered is from about 5 mg to about 40 mg.

42) The method of any one of exemplary embodiments 1 to 41, wherein theamount of the TLR agonist is less than an amount that is effective whenthe TLR agonist is administered systemically.

43) The method of any one of the preceding exemplary embodiments,comprising administering (i) an amount of the TLR agonist that is aninduction dose.

44) The method of exemplary embodiment 43, further comprising (ii)administering an amount of the TLR agonist that is a maintenance dosefollowing the administration of the induction dose.

45) The method of exemplary embodiment 43 or 44, wherein the inductiondose is administered once a day.

46) The method of exemplary embodiment 43 or 44, wherein the inductiondose is administered once every three days.

47) The method of exemplary embodiment 43 or 44, wherein the inductiondose is administered once a week.

48) The method of exemplary embodiment 44, wherein step (ii) is repeatedone or more times.

49) The method of exemplary embodiment 44, wherein the induction dose isequal to the maintenance dose.

50) The method of exemplary embodiment 44, wherein the induction dose isgreater than the maintenance dose.

51) The method of exemplary embodiment 44, wherein the induction dose is5 greater than the maintenance dose.

52) The method of exemplary embodiment 44, wherein the induction dose is2 greater than the maintenance dose.

53) The method of any one of the preceding exemplary embodiments,wherein the method comprises delivering the TLR agonist at the locationin the gastrointestinal tract as a single bolus.

54) The method of any one of exemplary embodiments 1 to 52, wherein themethod comprises delivering the TLR agonist at the location in thegastrointestinal tract as more than one bolus.

55) The method of any one of exemplary embodiments 1 to 52, wherein themethod comprises delivering the TLR agonist at the location in thegastrointestinal tract in a continuous manner.

56) The method of exemplary embodiment 55, wherein the method comprisesdelivering the TLR agonist at the location in the gastrointestinal tractover a time period of 20 or more minutes.

57) The method of any one of the preceding exemplary embodiments,wherein the method provides a concentration of the TLR agonist in theplasma of the subject that is less than 3 ng/ml.

58) The method of exemplary embodiment 57, wherein the method provides aconcentration of the TLR agonist in the plasma of the subject that isless than 0.3 ng/ml.

59) The method of exemplary embodiment 58, wherein the method provides aconcentration of the TLR agonist in the plasma of the subject that isless than 0.01 ng/ml.

60) The method of any one of exemplary embodiments 1 to 59, wherein themethod does not comprise delivering a TLR agonist rectally to thesubject.

61) The method of any one of exemplary embodiments 1 to 59, wherein themethod does not comprise delivering a TLR agonist via an enema to thesubject.

62) The method of any one of exemplary embodiments 1 to 59, wherein themethod does not comprise delivering a TLR agonist via suppository to thesubject.

63) The method of any one of exemplary embodiments 1 to 59, wherein themethod does not comprise delivering a TLR agonist via instillation tothe rectum of the subject.

64) The method of exemplary embodiment 63, wherein the TLR9 agonist is aTLR9 agonist.

65) The method of exemplary embodiment 63, wherein the TLR9 agonist isselected from cobitolimod (Kappaproct® or DIMS0150, InDexPharmaceuticals); generic equivalents thereof; modifications thereofhaving at least 90% sequence homology; modifications thereof having atleast 90% sequence homology; and modifications thereof having one ormore nucleotide insertions, deletions, or modifications.

66) The method of any one of the preceding exemplary embodiments,wherein the pharmaceutical composition is an ingestible device,comprising:

-   -   a housing defined by a first end, a second end substantially        opposite from the first end, and a wall extending longitudinally        from the first end to the second end;

a storage reservoir located within the housing and containing the TLRagonist,

wherein a first end of the storage reservoir is connected to the firstend of the housing;

a mechanism for releasing the TLR agonist from the storage reservoir;

and;

an exit valve configured to allow the TLR agonist to be released out ofthe housing from the storage reservoir.

67) The method of exemplary embodiment 66, wherein the ingestible devicefurther comprises:

an electronic component located within the housing; and

-   -   a gas generating cell located within the housing and adjacent to        the electronic component,    -   wherein the electronic component is configured to activate the        gas generating cell to generate gas.

68) The method of exemplary embodiment 66 or 67, wherein the ingestibledevice further comprises:

a safety device placed within or attached to the housing,

wherein the safety device is configured to relieve an internal pressurewithin the housing when the internal pressure exceeds a threshold level.

69) The method of exemplary embodiment 66, wherein the pharmaceuticalcomposition is an ingestible device, comprising:

-   -   a housing defined by a first end, a second end substantially        opposite from the first end, and a wall extending longitudinally        from the first end to the second end;    -   an electronic component located within the housing;    -   a gas generating cell located within the housing and adjacent to        the electronic component,        -   wherein the electronic component is configured to activate            the gas generating cell to generate gas;    -   a storage reservoir located within the housing,        -   wherein the storage reservoir stores a dispensable substance            and a first end of the storage reservoir is connected to the            first end of the housing;    -   an exit valve located at the first end of the housing,        -   wherein the exit valve is configured to allow the            dispensable substance to be released out of the first end of            the housing from the storage reservoir; and    -   a safety device placed within or attached to the housing,        -   wherein the safety device is configured to relieve an            internal pressure within the housing when the internal            pressure exceeds a threshold level.

70) The method of exemplary embodiment 66, wherein the pharmaceuticalcomposition is an ingestible device, comprising:

-   -   a housing defined by a first end, a second end substantially        opposite from the first end, and a wall extending longitudinally        from the first end to the second end;    -   an electronic component located within the housing,    -   a gas generating cell located within the housing and adjacent to        the electronic component,        -   wherein the electronic component is configured to activate            the gas generating cell to generate gas;    -   a storage reservoir located within the housing,        -   wherein the storage reservoir stores a dispensable substance            and a first end of the storage reservoir is connected to the            first end of the housing;    -   an injection device located at the first end of the housing,        -   wherein the jet injection device is configured to inject the            dispensable substance out of the housing from the storage            reservoir; and    -   a safety device placed within or attached to the housing,        -   wherein the safety device is configured to relieve an            internal pressure within the housing.

71) The method of exemplary embodiment 66, wherein the pharmaceuticalcomposition is an ingestible device, comprising:

-   -   a housing defined by a first end, a second end substantially        opposite from the first end, and a wall extending longitudinally        from the first end to the second end;

an optical sensing unit located on a side of the housing,

-   -   wherein the optical sensing unit is configured to detect a        reflectance from an environment external to the housing;

an electronic component located within the housing;

a gas generating cell located within the housing and adjacent to theelectronic component,

-   -   wherein the electronic component is configured to activate the        gas generating cell to generate gas in response to identifying a        location of the ingestible device based on the reflectance;    -   a storage reservoir located within the housing,        -   wherein the storage reservoir stores a dispensable substance            and a first end of the storage reservoir is connected to the            first end of the housing;    -   a membrane in contact with the gas generating cell and        configured to move or deform into the storage reservoir by a        pressure generated by the gas generating cell; and    -   a dispensing outlet placed at the first end of the housing,        -   wherein the dispensing outlet is configured to deliver the            dispensable substance out of the housing from the storage            reservoir.

72) The method of any one of exemplary embodiments 1-71, wherein thepharmaceutical composition is an ingestible device as disclosed in U.S.Patent Application Ser. No. 62/385,553, incorporated by reference hereinin its entirety.

73) The method of any one of exemplary embodiments 1-71, wherein thepharmaceutical composition is an ingestible device comprising alocalization mechanism as disclosed in international patent applicationPCT/US2015/052500, incorporated by reference herein in its entirety.

74) The method of any one of exemplary embodiments 1-73, wherein thepharmaceutical composition is not a dart-like dosage form.

75) A method of treating a disease of the large intestine of a subject,comprising: delivering of a TLR agonist at a location in the proximalportion of the large intestine of the subject, wherein the methodcomprises administering endoscopically to the subject a therapeuticallyeffective amount of the TLR agonist.

76) The method of exemplary embodiment 75, wherein the disease of thelarge intestine is an inflammatory bowel disease.

77) The method of exemplary embodiment 75, wherein the disease of thelarge intestine is ulcerative colitis.

78) The method of exemplary embodiment 75, wherein the disease the largeintestine is Crohn's disease.

79) The method of any one of exemplary embodiments 75 to 78, wherein theTLR agonist is delivered at a location in the proximal portion of theascending colon.

80) The method of any one of exemplary embodiments 75 to 78, wherein theTLR agonist is delivered at a location in the proximal portion of thececum.

81) The method of any one of exemplary embodiments 75 to 78, wherein theTLR agonist is delivered at a location in the proximal portion of thesigmoid colon.

82) The method of any one of exemplary embodiments 75 to 78, wherein theTLR agonist is delivered at a location in the proximal portion of thetransverse colon.

83) The method of any one of exemplary embodiments 75 to 78, wherein theTLR agonist is delivered at a location in the proximal portion of thedescending colon.

84) The method of any one of the preceding exemplary embodiments,further comprising administering a second agent orally, intravenously orsubcutaneously, wherein the second agent is the same TLR agonist as inexemplary embodiment 1 or 75; a different TLR agonist; or an agenthaving a different biological target from TLR.

85) The method of any one of the preceding exemplary embodiments,further comprising administering a second agent orally, intravenously orsubcutaneously, wherein the second agent is an agent suitable fortreating an inflammatory bowel disease.

86) The method of exemplary embodiment 84 or 85, wherein the TLR agonistis administered prior to the second agent.

87) The method of exemplary embodiment 84 or 85, wherein the TLR agonistis administered after the second agent.

88) The method of exemplary embodiment 84 or 85, wherein the TLR agonistand the second agent are administered substantially at the same time.

89) The method of any one of exemplary embodiments 84 to 88, wherein thesecond agent is administered intravenously.

90) The method of any one of exemplary embodiments 84 to 88, wherein thesecond agent is administered subcutaneously.

91) The method of any one of exemplary embodiments 84 to 90, wherein theamount of the second agent is less than the amount of the second agentwhen the TLR agonist and the second agent are both administeredsystemically.

92) The method of exemplary embodiment 91, wherein the second agent isan immunosuppressant.

93) In some aspects of these embodiments, the second agent ismethotrexate.

94) The method of any one of exemplary embodiments 1 to 83, wherein themethod does not comprise administering a second agent.

Other Embodiments

The various embodiments of systems, processes and apparatuses have beendescribed herein by way of example only. It is contemplated that thefeatures and limitations described in any one embodiment may be appliedto any other embodiment herein, and flowcharts or examples relating toone embodiment may be combined with any other embodiment in a suitablemanner, done in different orders, or done in parallel. It should benoted, the systems and/or methods described above may be applied to, orused in accordance with, other systems and/or methods. Variousmodifications and variations may be made to these example embodimentswithout departing from the spirit and scope of the embodiments, and theappended listing of embodiments should be given the broadestinterpretation consistent with the description as a whole.

The invention claimed is:
 1. A method of treating ulcerative colitis ina subject, the method comprising: orally administering to the subject aningestible device comprising: an ingestible housing comprising areservoir, the reservoir containing a pharmaceutical formulationcomprising a therapeutically effective amount of a TLR modulator; arelease mechanism having a closed state wherein the pharmaceuticalformulation is retained in the reservoir and an open state which allowsfor release of the pharmaceutical formulation from the reservoir to anexterior of the ingestible device; an actuator which controls atransition of the release mechanism from the closed state to the openstate; a light source configured to produce light that interacts with agastrointestinal (GI) tract of the subject to provide light reflectance;a detector configured to detect the light reflectance to detect the (GI)tract; and a processor coupled to the detector and to the actuator,wherein the processor triggers the actuator to cause the releasemechanism to transition from the closed state to the open state when theingestible device is located in the cecum based on the detected lightreflectance, wherein the cecum has been predetermined to be proximal toone or more disease sites, thereby releasing the pharmaceuticalformulation comprising the TLR modulator from the ingestible device whenthe ingestible device is located in the cecum of the subject.
 2. Themethod of claim 1, wherein the one or more disease sites is in thecolon.
 3. The method of claim 1, wherein the TLR modulator is a TLRagonist.
 4. The method of claim 3, wherein the TLR agonist is a TLR9agonist selected from the group consisting of cobitolimod; genericequivalents thereof; modifications thereof having at least 90% sequencehomology; and modifications thereof having one or more nucleotideinsertions, deletions, or modifications.
 5. The method of claim 1,wherein the ingestible device further comprises one or moremachine-readable hardware storage devices that stores instructions thatare executable by the processor to determine that the ingestible deviceis in the cecum of the subject to an accuracy of at least 70%.
 6. Themethod of claim 1, wherein the method further comprises determining thelocation of the ingestible device in the cecum of the subject to anaccuracy of at least 85%.
 7. The method of claim 1, wherein the detectedreflectance autonomously triggers the release of the pharmaceuticalformulation comprising the TLR modulator from the ingestible device. 8.The method of claim 1, wherein the detected reflectance comprises lightof at least two different wavelengths.
 9. The method of claim 1, whereindetermining the location of the ingestible device in the cecum comprisesdetecting a transition of the ingestible device from the ileum to thececum.
 10. The method of claim 9, wherein detecting the transition ofthe ingestible device from the ileum to the cecum comprises detecting achange in a ratio of reflected red light to reflected green light. 11.The method of claim 10, wherein detecting the transition of theingestible device from the ileum to the cecum further comprisesdetecting a change in a ratio of reflected green light to reflected bluelight.
 12. The method of claim 1, wherein the ingestible devicecomprises a gas generating cell located within the housing, wherein thegas generating cell is capable of generating a gas; and the ingestibledevice is configured so that, when the gas generating cell generates thegas, the gas creates an internal pressure that forces the releasemechanism from the closed state, which retains the TLR modulator in thereservoir, to the open state, thereby allowing for the release of theTLR modulator from the reservoir to the exterior of the device.
 13. Themethod of claim 12, wherein the reservoir is configured to friction fitwith the ingestible device.
 14. The method of claim 12, wherein thereservoir is configured to attach to the housing of the ingestibledevice.
 15. The method of claim 12, wherein the ingestible devicecomprises a safety device placed within or attached to the housing,wherein the safety device is configured to relieve the internal pressurewithin the housing when the internal pressure exceeds a threshold level.16. The method of claim 1, further comprising releasing thepharmaceutical formulation comprising the TLR modulator to the cecum asa bolus.
 17. The method of claim 1, further comprising determining alevel of TLR modulator in the plasma of the subject following the oraladministration of the ingestible device, wherein the level of TLRmodulator in the plasma of the subject is lower than the level of theTLR modulator in the plasma of the subject at substantially the sametime point following systemic administration of an equal amount of theTLR modulator.
 18. The method of claim 1, wherein releasing the TLRmodulator from the ingestible device is not dependent on pH, enzymaticactivity or bacterial activity at or in the vicinity of thepredetermined location.